Use of anti-pacap antibodies and antigen binding fragments thereof for treatment, prevention, or inhibition of photophobia

ABSTRACT

This invention relates to methods of screening for anti-PACAP antibodies, or anti-PACAP receptor antibodies, and antigen binding fragments thereof, for potential use in treating or preventing PACAP-associated photophobia or light aversion, and therapeutic compositions containing and methods of using anti-PACAP antibodies, or anti-PACAP receptor antibodies, and antigen binding fragments thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/148,550, filed Apr. 16, 2015, U.S. Provisional Application Ser.No. 62/148,557, filed Apr. 16, 2015, U.S. Provisional Application Ser.No. 62/148,562, filed Apr. 16, 2015, U.S. Provisional Application Ser.No. 62/148,596, filed Apr. 16, 2015, U.S. Provisional Application Ser.No. 62/148,643, filed Apr. 16, 2015, U.S. Provisional Application Ser.No. 62/148,583, filed Apr. 16, 2015, U.S. Provisional Application Ser.No. 62/148,640, filed Apr. 16, 2015, each of which is herebyincorporated by reference in its entirety.

SEQUENCE DISCLOSURE

This application includes as part of its disclosure an electronicsequence listing text file named “43257o6400.txt” having a size of478,737 bytes and created Aug. 26, 2016, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention generally pertains to in vivo screening assays foridentification of agents able to treat or prevent photophobia, as wellas antibodies and antigen binding fragments thereof, preferablyhumanized, chimerized, and human antibodies and antigen bindingfragments thereof, and compositions containing such antibodies andantigen binding fragments thereof, wherein such antibodies and antigenbinding fragments thereof specifically bind to Pituitary AdenylateCyclase-Activating Polypeptide (“PACAP”) and therapeutic and diagnosticuses for the antibodies, antigen binding fragments, and compositionsthereof.

BACKGROUND

Pituitary Adenylate Cyclase-Activating Polypeptide (“PACAP”) is a memberof the secretin/vasoactive intestinal peptide (“VIP”)/growthhormone-releasing hormone (“GHRH”) family. PACAP is a multifunctionalvasodilatory peptide that exists in two α-amidated active forms, onewith 38 amino acids (PACAP38; SEQ ID NO: 1241) and the other with 27amino acids (PACAP27; SEQ ID NO: 1242). Both peptides have the sameN-terminal 27 amino acids and are synthesized from the same precursorprotein, preproPACAP (See, Moody et al., Curr. Opin. Endocrinol.Diabetes Obes., 18(1): 61-67, 2011). PACAP38 is the more prevalentactive form, representing up to 90% of PACAP forms in mammalian tissues(See, Kaiser and Russo, Neuropeptides, 47:451-461, 2013). The sequenceof PACAP38 is identical in all mammals and differs from the avian andamphibian orthologs by only one amino acid (See, Vaudry et al.,Pharmacol. Rev., 52:269-324, 2000). The secretin/VIP/GHRH familyincludes mammalian peptide histidine methioneamide (“PHM”), secretin,glucagon, glucagon-like peptide-1 (“GLP1”), glucagon-like peptide-2(“GLP2”), glucose-dependent-insulinotrophic-polypeptide (“GIP”), andgrowth-hormone-releasing-factor (“GRF”). PACAP27 has 68% sequenceidentity to VIP at the amino acid level (See, Vaudry et al. 2000).

PACAP is widely distributed in the brain and peripheral organs, e.g.,the endocrine system, gonads, sympathetic neurons, respiratory system,gastrointestinal tract, cardiovascular system, and urogenital tracts(See, Schytz et al., Neurotherapeutics, 7:191-196, 2010). In particular,PACAP is expressed throughout the nervous system, including a presencein the trigeminovascular system, trigeminal ganglia, spinal cord,hypothalamus, and pituitary. PACAP has roles in neurodevelopment,neuroprotection, neuromodulation, neurogenic inflammation, andnociception with multiple actions (See, Kaiser and Russo (2013)).

Consistent with its widespread distribution, PACAP exerts pleiotropiceffects including modulation of neurotransmitter release, vasodilation,bronchodilation, and activation of intestinal motility, increase ofinsulin and histamine secretion, as well as stimulation of cellproliferation and/or differentiation. PACAP has been shown to act as ahormone, a neurohormone, a neurotransmitter, and a trophic factor in anumber of tissues (See, Vaudry et al., Pharmacological Rev.,52(2):269-324, 2000).

The biological effects of PACAP are mediated via three differentG-protein coupled receptors: PAC1-R, vasoactive intestinal peptidereceptor type 1 (“VPAC1-R”), and vasoactive intestinal peptide receptortype 2 (“VPAC2-R”). These receptors are expressed in diverse tissues.PAC1-R is particularly abundant in the nervous system (e.g., olfactorybulb, thalamus, hypothalamus, cerebellum, and spinal dorsal horn),pituitary, and adrenal glands. By contrast, VPAC1-R and VPAC2-R areexpressed mainly in the lung, liver, and testis, although they have beendetected in other tissues as well. VPAC1-R expression has been detectedin the nervous system (e.g., cerebral cortex and hippocampus), smoothmuscle cells of lung, liver, intestine, megakaryocytes, and platelets.VPAC1-R associates with receptor-associated membrane protein (“RAMP”,specifically RAMP2) (See, Christopoulos et al., J. Biol. Chem.,278:3293-3297, 2002). VPAC2-R expression profile includes the nervous(e.g., thalamus, hippocampus, brain stem, and dorsal root ganglia(“DRG”)), cardiovascular system, gastrointestinal system, pancreas, andreproductive systems (See, Usdin et al., Endocrin., 135:2662-2680, 1994;Sheward et al., Neurosci., 67:409-418, 1995).

PAC1-R is selective for PACAP38 and PACAP27. In particular, PAC1-R bindsto PACAP with 100-1000-fold greater affinity than VIP, i.e., K_(D)˜0.5nM for PACAP27/PACAP38 vs. K_(D)˜500 nM for VIP. Conversely, VPAC1-R andVPAC2-R have equal affinities for PACAP and VIP (K_(D)˜1 nM) (See Schytzet al. (2010)).

Upon activation, these receptors are all capable of causing downstreamproduction of cyclic adenosine monophosphate (“cAMP”), and/or activationof phospholipase C (“PLC”), and/or modulation of phospholipase D(“PLD”). In particular, PAC1-R is coupled to dual signal transductionpathways acting through cAMP and Ca²⁺, whereas VPAC1-R and VPAC2-R arecoupled principally to adenylyl cyclase. PAC1-R is coupled to G_(s)protein, which activates adenylyl cyclase to form cAMP that in turnactivates protein kinase A. PAC1-R also couples to Gq and therebyactivates PLC, which produces inositol phosphate, which increasescytosolic calcium release from intra-cellular calcium stores. There issome evidence for a role of PAC1-R in PLD activation (See McCulloch etal., Ann. N. Y. Acad. Sci., 921:175-185, 2000). Another PACAP signalingpathway results in the elevation of intra-cellular sodium levels viaactivation of nonselective cation channels (See Roy et al., AmericanJournal of Physiology: Regulatory, Integrative and ComparativePhysiology, 304(12):R1070-R1084, 2013).

PACAP is hypothesized to play a role in a multitude of diseases anddisorders, including but not limited to migraine, headache, and pain,though such a role for PACAP has not been clinically demonstrated.Migraines are believed to have a neurovascular component. Migrainesaffect approximately 10% of the adult population in the U.S. and aretypically accompanied by intense headaches. Approximately 20-30% ofmigraine sufferers experience aura, comprising focal neurologicalphenomena that precede and/or accompany the event. A role for PACAP inmigraine has been suggested by several observations: (1) plasma levelsof PACAP are elevated during migraine attacks (ictal), as compared tointerictal levels, in humans (see, Tuka et al., Cephalalgia,33(13):1085-1095, 2013); (2) an infusion of PACAP38 triggered headachesin healthy subjects, and headaches followed by migraine-like attacks inmigraineurs (see, Schytz et al., Brain, 132:16-25 2009; and Amin et al.,Brain, 137:779-794, 2014, respectively); (3) PACAP-induced vasodilationmay play a role in neurogenic inflammation (see, Kaiser and Russo,Neuropeptides, 47:451-461, 2013); and (4) PACAP-induced migraines areassociated with photophobia, phonophobia, nausea, and respond totriptans (see, Amin et al., Brain, 32:140-149 2012). PACAP has also beenshown to induce vasodilation, photophobia, as well as mast celldegranulation and neuronal activation (See, Markovics et al.,Neurobiology of Disease, 45:633-644 2012; Baun et al., Cephalalgia,32(4):337-345, 2012; Chan et al., Pharmacology & Therapeutics,129:332-351, 2011).

One effective treatment for migraines is the administration of triptans,which are a family of tryptamine-based drugs, including sumatriptan andrizatriptan. Members of this family have an affinity for multipleserotonin receptors, including 5-HT_(1B), 5-HT_(1D), and 5-HT_(1F).Members of this family of drugs selectively constrict cerebral vessels,but also cause vasoconstrictive effects on coronary vessels (See Durham,New Eng. J. Med., 350 (11):1073-75, 2004). There is a theoretical riskof coronary spasm in patients with established heart disease followingadministration, and cardiac events after taking triptans in rareinstances may occur. Accordingly, they are contraindicated for somepatients with coronary vascular disease.

Similarly, pain may often be addressed through the administration ofcertain narcotics or non-steroidal anti-inflammatory drugs (“NSAIDs”).However, the administration of these treatments often has negativeconsequences. NSAIDs have the potential to cause kidney failure,intestinal bleeding, and liver dysfunction. Narcotics have the potentialto cause nausea, vomiting, impaired mental functioning, and addiction.Therefore, it is desirable to identify alternative treatments for painin order to avoid certain of these negative consequences.

PACAP may also be involved in diseases and disorders other thanmigraine, headache, and pain. For example, PACAP may correlate to oreven play a causal role in anxiety disorders (WO 2012/106407);thrombocytopenia (WO 2004/062684); and inflammatory skin diseases (WO2010/007175). PACAP and PAC1-R polymorphisms are associated withpost-traumatic stress syndrome (“PTSD”) in females, major depressivedisorder, and generalized anxiety disorder, suggesting a role for PACAPin these conditions. Further, supporting a role for PACAP inthrombocytopenia, trisomy 18 patients have excess PACAP and exhibitdefective megakaryocyte maturation (See, Schytz et al. 2010; and Moodyet al., Curr. Opin. Endocrinol. Diabetes Obes., 18(1):61-67, 2011).

Also, PACAP and other neuropeptides, such as Calcitonin Gene-RelatedPeptide (“CGRP”), substance P, neurokinin A, bradykinin, andendothelin-1, are expressed in the lower urinary tract (“LUT”) (see,Arms and Vizzard, Handbook Exp. Pharmacol., 202:395-423 2011) andreportedly may play a role in LUT dysfunction and urinary tractdisorders such as urinary tract infection (“UTI”), abnormal voiding,urinary urgency, nocturia, urinary incontinence, overactive bladder, andthe pain associated with such conditions.

PACAP and PACAP receptors have also been suggested to modulateinflammatory and neuropathic pain and have been implicated in bothpronociception and antinociception (See, Davis-Taber et al., J. Pain,9(5):449-56 2008). PACAP has also been reported to be required forspinal desensitization and the induction of neuropathic pain (See,Mabuchi et al., J. Neurosci., 24(33):7283-91, 2004). Additionally,morphine withdrawal behavior is reportedly modified in PACAP-receptordeficient mice further suggesting the role of PACAP in morphinewithdrawal anxiolytic response (See, Martin et al., Mol. Brain Res.,110(1):109-18 2003).

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention in general relates to a method ofscreening for an antibody or antigen binding fragment that may besuitable for use in treating or preventing Pituitary AdenylateCyclase-Activating Peptide (PACAP)-associated photophobia or lightaversion, or precluding the onset of PACAP-associated photophobia orlight aversion, in a subject in need thereof, which may comprise:

-   -   (i) providing at least one first test subject and at least one        second test subject;    -   (ii) administering PACAP to the at least one first test subject        and the at least one second test subject;    -   (iii) further administering to the at least one first test        subject one or more anti-PACAP antibodies, anti-Pituitary        Adenylate Cyclase Activating Polypeptide type 1 Receptor        (PAC1-R) antibodies, anti-vasoactive intestinal peptide receptor        type 1 (VPAC1-R) antibodies, or anti-vasoactive intestinal        peptide receptor type 2 (VPAC2-R) antibodies, or an antigen        binding fragment of any of the foregoing;    -   (iv) comparing the response of the at least one first test        subject and at least one second test subject to light; and    -   (v) based on this comparison, identifying one or more antibodies        or antigen binding fragments thereof that yield a decreased        light aversion or decreased photophobia in the at least one        first test subject as compared with the at least one second test        subject, thereby identifying an antibody or antigen binding        fragment suitable for use in treating or preventing        PACAP-associated photophobia or light aversion, or precluding        the onset of PACAP-associated photophobia or light aversion in a        subject in need thereof.

In certain embodiments, the invention embraces a method of administeringone or more antibodies or antigen binding fragments may compriseadministering at least one anti-PACAP antibody or antigen bindingfragment thereof. In another embodiment, the invention relates to amethod of administering one or more antibodies or antigen bindingfragments that may comprise administering at least one anti-PAC1-Rantibody or antigen binding fragment thereof. The invention may alsogenerally pertain to a method wherein the efficacy of the one or moreantibodies or antigen binding fragments for inhibiting PACAP-associatedphotophobia or light aversion, or precluding the onset ofPACAP-associated photophobia or light aversion, may be confirmed in ahuman subject. Moreover, another aspect of the invention relates to amethod wherein at least one first test subject and at least one secondtest subject may be different subjects.

Furthermore, the invention generally embraces a method of screening foran antibody or antigen binding fragment thereof that may be suitable foruse in treating or preventing Pituitary Adenylate Cyclase-ActivatingPeptide (PACAP)-associated photophobia or light aversion, or precludingthe onset of PACAP-associated photophobia or light aversion, in asubject in need thereof, which may comprise:

-   -   (i) providing at least one first test subject and at least one        second test subject;    -   (ii) administering to the at least one first test subject one or        more anti-PACAP antibodies, anti-Pituitary Adenylate Cyclase        Activating Polypeptide type 1 Receptor (PAC1-R) antibodies,        anti-vasoactive intestinal peptide receptor type 1 (VPAC1-R)        antibodies, or anti-vasoactive intestinal peptide receptor type        2 (VPAC2-R) antibodies, preferably at least one anti-PACAP        antibody and/or at least one anti-PAC1-R antibody, or an antigen        binding fragment of any of the foregoing;    -   (iii) administering PACAP to the at least one first test subject        and the at least one second test subject;    -   (iv) comparing the response of the at least one first test        subject and at least one second test subject to light; and    -   (v) based on this comparison, identifying antibodies or antigen        binding fragments that yield decreased photophobia or decreased        light aversion in the at least one first test subject as        compared with the at least one second test subject, identifying        an antibody or antigen binding fragment suitable for use in        treating or preventing PACAP-associated photophobia or light        aversion, or identifying an antibody or antigen binding fragment        suitable for precluding the onset of PACAP-associated        photophobia or light aversion, in a subject in need thereof.

Moreover, the invention relates to a method wherein administering one ormore antibody or antigen binding fragments may comprise administeringone or more anti-PACAP antibodies or antigen binding fragments thereof.Furthermore, one aspect of the invention generally pertains to a methodwherein administering the one or more antibody or antigen bindingfragments may comprise administering one or more anti-PAC1-R antibodiesor antigen binding fragments thereof. In another embodiment, theinvention embraces a method wherein the efficacy of one or moreantibodies or antigen binding fragments for inhibiting PACAP-associatedphotophobia or light aversion, or precluding the onset ofPACAP-associated photophobia or light aversion, may be confirmed in ahuman subject. Additionally, the invention relates to a method whereinthe at least one first test subject and/or the at least one second testsubject of any of the methods disclosed herein may be a mammal, e.g.,wherein the at least one first test subject and/or at least one secondtest subject may be a mouse, monkey, rabbit, human, rat, guinea pig,dog, or hamster. Furthermore, said monkey may be a macaque, marmoset,tamarin, spider monkey, owl money, vervet monkey, squirrel monkey, orbaboon. Additionally, the invention generally relates to a methodwherein the at least one first test subject of any of the methodsdisclosed herein or at least one second test subject of any of themethods disclosed herein may be a mouse, e.g., a CD1 mouse. Theinvention also embraces any of the methods disclosed herein wherein theidentified antibody or antigen binding fragment thereof may specificallybind PACAP.

In an additional embodiment, the invention relates to a method that maycomprise adapting the identified antibody or antigen binding fragmentfor use in treating a subject who may suffer from one or more ofmigraine, hemiplegic migraines, cluster headaches, migrainous neuralgia,chronic headaches, tension headaches, secondary headaches due to anunderlying structural problem in the head or neck, cranial neuralgia,sinus headaches, allergy-induced headaches, headache, or other migrainecondition. Additionally, said subject of any of the methods disclosedherein may have an ocular disorder associated with photophobia that maybe selected from one or more of achromatopsia, aniridia, photophobiacaused by an anticholinergic drug, aphakia (absence of the lens of theeye), buphthalmos (abnormally narrow angle between the cornea and iris),cataracts, cone dystrophy, congenital abnormalities of the eye, viralconjunctivitis (“pink eye”), corneal abrasion, corneal dystrophy,corneal ulcer, disruption of the corneal epithelium, ectopia lentis,endophthalmitis, eye trauma caused by disease, injury, or infection suchas chalazion, episcleritis, glaucoma, keratoconus, or optic nervehypoplasia, hydrophthalmos, or congenital glaucoma Iritis, opticneuritis, pigment dispersion syndrome, pupillary dilation (naturally orchemically induced), retinal detachment, scarring of the cornea orsclera, and uveitis. Also, said subject of any of the methods disclosedherein may have a nervous-system-related or neurological conditionassociated with photophobia selected from one or more of autism spectrumdisorders, chiari malformation, dyslexia, encephalitis including myalgicencephalomyelitis (chronic fatigue syndrome), meningitis, subarachnoidhemorrhage, tumor of the posterior cranial fossa, ankylosingspondylitis, albinism, ariboflavinosis, benzodiazepines (long term useof or withdrawal from benzodiazepines), chemotherapy, chikungunyainfection, cystinosis, Ehlers-Danlos syndrome, hangover, influenzainfection, infectious mononucleosis, magnesium deficiency, mercurypoisoning, migraine, rabies, and tyrosinemia type II (Richner-Hanhartsyndrome). Furthermore, said subject of any of the methods disclosedherein may have a photophobia associated disorder that may be selectedfrom one or more of migraine (with or without aura), iritis, uveitis,meningitis, depression, bipolar disorder, cluster headache or anothertrigeminal autonomic cephalalgia or blepharospasm, depression,post-traumatic stress syndrome (PTSD) traumatic brain injury, andagoraphobia. In a further embodiment, the invention relates to a methodwherein the at least one first subject and/or at least one secondsubject may suffer from migraine headaches. Yet another embodiment ofthe invention relates to a method wherein the one or more antibodies orantigen binding fragments thereof may be for use in combination withanother active agent for treating migraine. Also, the inventiongenerally pertains to a method wherein the identified antibody orantigen binding fragment thereof may be for use as a monotherapy. Forexample, said antibody or antigen binding fragment thereof may be foruse in combination with another active agent effective for treatingmigraine, wherein antibody and active agent may be for joint or separateadministration. Also, said antibody or fragment thereof may be for usein treating subjects suffering from migraines, in combination with oneor more of an analgesic, a triptan, a topiramate, a dihydroergotamine,and an opioid.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies or antigen binding fragments thereof identified inthe disclosed methods, comprise human, humanized, or chimerizedanti-PACAP antibodies or antigen binding fragments thereof, whichcomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 4; a CDR2 sequence consisting of SEQ ID NO: 6;and a CDR3 sequence consisting of SEQ ID NO: 8; and/or (b) a variablelight chain comprising a CDR1 sequence consisting of SEQ ID NO: 24; aCDR2 sequence consisting of SEQ ID NO: 26; and a CDR3 sequenceconsisting of SEQ ID NO: 28. Alternatively, the selected anti-PACAPantibodies or antigen binding fragments may comprise (a) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 2 and/or (b) avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 22. Inanother specific embodiment, the selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 2, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO:22. Inanother embodiment, the selected anti-PACAP antibodies or antigenbinding fragments comprise (a) a heavy chain having the amino acidsequence of SEQ ID NO: 1, and/or (b) a light chain having the amino acidsequence of SEQ ID NO: 21.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments, comprise (a) avariable heavy chain comprising a CDR1 sequence consisting of SEQ ID NO:44; a CDR2 sequence consisting of SEQ ID NO: 46; and a CDR3 sequenceconsisting of SEQ ID NO: 48; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO: 64; a CDR2 sequenceconsisting of SEQ ID NO: 66; and a CDR3 sequence consisting of SEQ IDNO: 68. More specifically, the selected anti-PACAP antibodies or antigenbinding fragments thereof comprise (a) a variable heavy chain comprisingan amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99%sequence identity to SEQ ID NO: 42, and/or (b) a variable light chaincomprising an amino acid sequence with at least 80, 85, 90, 95, 96, 97,98, or 99% sequence identity to SEQ ID NO: 62. In another specificembodiment, the selected anti-PACAP antibodies or antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 42, and/or (b) a variable light chain havingthe amino acid sequence of SEQ ID NO: 62. More specifically, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 41,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:61.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments, may comprise (a) avariable heavy chain comprising a CDR1 sequence consisting of SEQ ID NO:84; a CDR2 sequence consisting of SEQ ID NO:86; and a CDR3 sequenceconsisting of SEQ ID NO: 88; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO: 104; a CDR2 sequenceconsisting of SEQ ID NO: 106; and a CDR3 sequence consisting of SEQ IDNO: 108. In a further specific embodiment, the selected anti-PACAPantibodies or antigen binding fragments comprise (a) a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 82, and/or (b) avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 102. Inanother embodiment, the selected anti-PACAP antibodies or antigenbinding fragments comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 82, and/or (b) a variable light chain havingthe amino acid sequence of SEQ ID NO: 102. In another specificembodiment, the selected anti-PACAP antibodies and antigen bindingfragments thereof may comprise (a) a heavy chain having the amino acidsequence of SEQ ID NO: 81, and/or (b) a light chain having the aminoacid sequence of SEQ ID NO: 101.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies or antigen binding fragments, may comprise (a) avariable heavy chain comprising a CDR1 sequence consisting of SEQ ID NO:124; a CDR2 sequence consisting of SEQ ID NO: 126; and a CDR3 sequenceconsisting of SEQ ID NO: 128; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO: 144; a CDR2 sequenceconsisting of SEQ ID NO:146; and a CDR3 sequence consisting of SEQ IDNO: 148. In a further specific embodiment, the selected anti-PACAPantibodies or antigen binding fragments may comprise (a) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 122 and/or (b) avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 142. Inanother specific embodiment, selected anti-PACAP antibodies and antigenbinding fragments thereof may comprise (a) a variable heavy chain havingthe amino acid sequence of SEQ ID NO: 122, and/or (b) a variable lightchain having the amino acid sequence of SEQ ID NO: 142. In anotherspecific embodiment, selected anti-PACAP antibodies and antigen bindingfragments thereof may comprise (a) a heavy chain having the amino acidsequence of SEQ ID NO: 121, and/or (b) a light chain having the aminoacid sequence of SEQ ID NO: 141.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments identified by thedisclosed methods, preferably human, humanized, or chimerized anti-PACAPantibodies and antigen binding fragments thereof, may comprise (a) avariable heavy chain comprising a CDR1 sequence consisting of SEQ ID NO:164; a CDR2 sequence consisting of SEQ ID NO: 166; and a CDR3 sequenceconsisting of SEQ ID NO: 168; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO: 184; a CDR2 sequenceconsisting of SEQ ID NO: 186; and a CDR3 sequence consisting of SEQ IDNO: 188. In a further specific embodiment, selected anti-PACAPantibodies and antigen binding fragments thereof may comprise (a) avariable heavy chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 162,and/or (b) a variable light chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 182. In another specific embodiment, selected anti-PACAP antibodiesand antigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 162, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 182.In a further specific embodiment, selected anti-PACAP antibodies orantigen binding fragments thereof may comprise (a) a heavy chain havingthe amino acid sequence of SEQ ID NO: 161, and/or (b) a light chainhaving the amino acid sequence of SEQ ID NO: 181.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 204; a CDR2 sequence consisting of SEQ ID NO:206; and a CDR3 sequence consisting of SEQ ID NO: 208; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:224; a CDR2 sequence consisting of SEQ ID NO: 226; and a CDR3 sequenceconsisting of SEQ ID NO: 228. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprise(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:202, and/or (b) a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 222. In another specific embodiment, selected anti-PACAPantibodies and antigen binding fragments thereof may comprise (a) avariable heavy chain having the amino acid sequence of SEQ ID NO: 202,and/or (b) a variable light chain having the amino acid sequence of SEQID NO: 222. In another specific embodiment, selected anti-PACAPantibodies and antigen binding fragments thereof may comprise (a) aheavy chain having the amino acid sequence of SEQ ID NO: 201, and/or (b)a light chain having the amino acid sequence of SEQ ID NO: 221.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof selected bythe disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 244; a CDR2 sequence consisting of SEQ ID NO:246; and a CDR3 sequence consisting of SEQ ID NO: 248; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:264; a CDR2 sequence consisting of SEQ ID NO: 266; and a CDR3 sequenceconsisting of SEQ ID NO: 268. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprise(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:242, and/or (b) a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 262. In another specific embodiment, selected anti-PACAPantibodies and antigen binding fragments thereof may comprise (a) avariable heavy chain having the amino acid sequence of SEQ ID NO: 242,and/or (b) a variable light chain having the amino acid sequence of SEQID NO: 262. In another specific embodiment, the selected anti-PACAPantibodies and antigen binding fragments thereof may comprise (a) aheavy chain having the amino acid sequence of SEQ ID NO: 241, and/or (b)a light chain having the amino acid sequence of SEQ ID NO: 261.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprising (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 284; a CDR2 sequence consisting of SEQ ID NO:286; and a CDR3 sequence consisting of SEQ ID NO: 288; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:304; a CDR2 sequence consisting of SEQ ID NO: 306; and a CDR3 sequenceconsisting of SEQ ID NO: 308. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprisea variable heavy chain comprising an amino acid sequence with at least80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 282,and/or a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:302. In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 282, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 302.In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a heavy chain havingthe amino acid sequence of SEQ ID NO: 281, and/or (b) a light chainhaving the amino acid sequence of SEQ ID NO: 301.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 324; a CDR2 sequence consisting of SEQ ID NO:326; and a CDR3 sequence consisting of SEQ ID NO: 328; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:344; a CDR2 sequence consisting of SEQ ID NO: 346; and a CDR3 sequenceconsisting of SEQ ID NO: 348. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprisea variable heavy chain comprising an amino acid sequence with at least80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 322,and/or a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:342. In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 322, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 342.In another specific embodiment, the selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a heavy chain havingthe amino acid sequence of SEQ ID NO: 321, and/or (b) a light chainhaving the amino acid sequence of SEQ ID NO: 341.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 364; a CDR2 sequence consisting of SEQ ID NO:366; and a CDR3 sequence consisting of SEQ ID NO: 368; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:384; a CDR2 sequence consisting of SEQ ID NO: 386; and a CDR3 sequenceconsisting of SEQ ID NO: 388. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprisea variable heavy chain comprising an amino acid sequence with at least80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 362,and/or a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:382. In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 362, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 382.In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a heavy chain havingthe amino acid sequence of SEQ ID NO: 361, and/or (b) a light chainhaving the amino acid sequence of SEQ ID NO: 381.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 484; a CDR2 sequence consisting of SEQ ID NO:486; and a CDR3 sequence consisting of SEQ ID NO: 488; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:504; a CDR2 sequence consisting of SEQ ID NO: 506; and a CDR3 sequenceconsisting of SEQ ID NO: 508. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprisea variable heavy chain comprising an amino acid sequence with at least80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 482,and/or a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:502. In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 482, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 502.In a specific embodiment, selected anti-PACAP antibodies and antigenbinding fragments thereof may comprise (a) a heavy chain having theamino acid sequence of SEQ ID NO: 481, and/or (b) a light chain havingthe amino acid sequence of SEQ ID NO: 501.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 524; a CDR2 sequence consisting of SEQ ID NO:526; and a CDR3 sequence consisting of SEQ ID NO: 528; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:544; a CDR2 sequence consisting of SEQ ID NO: 546; and a CDR3 sequenceconsisting of SEQ ID NO: 548. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprisea variable heavy chain comprising an amino acid sequence with at least80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 522,and/or a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:542. In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 522, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 542.In a further specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a heavy chain havingthe amino acid sequence of SEQ ID NO: 521, and/or (b) a light chainhaving the amino acid sequence of SEQ ID NO: 541.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 564; a CDR2 sequence consisting of SEQ ID NO:566; and a CDR3 sequence consisting of SEQ ID NO: 568; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:584; a CDR2 sequence consisting of SEQ ID NO: 586; and a CDR3 sequenceconsisting of SEQ ID NO: 588. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprisea variable heavy chain comprising an amino acid sequence with at least80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 562,and/or a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:582. In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 562, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 582.In a further specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a heavy chain havingthe amino acid sequence of SEQ ID NO: 561, and/or (b) a light chainhaving the amino acid sequence of SEQ ID NO: 581.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 604; a CDR2 sequence consisting of SEQ ID NO:606; and a CDR3 sequence consisting of SEQ ID NO: 608; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:624; a CDR2 sequence consisting of SEQ ID NO: 626; and a CDR3 sequenceconsisting of SEQ ID NO: 628. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprisea variable heavy chain comprising an amino acid sequence with at least80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 602,and/or a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:622. In a further specific embodiment, selected anti-PACAP antibodiesand antigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 602, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 622.In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a heavy chain havingthe amino acid sequence of SEQ ID NO: 601, and/or (b) a light chainhaving the amino acid sequence of SEQ ID NO: 621.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 644; a CDR2 sequence consisting of SEQ ID NO:646; and a CDR3 sequence consisting of SEQ ID NO: 648; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:664; a CDR2 sequence consisting of SEQ ID NO: 666; and a CDR3 sequenceconsisting of SEQ ID NO: 668. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprisea variable heavy chain comprising an amino acid sequence with at least80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 642,and/or a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:662. In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 642, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 662.In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a heavy chain havingthe amino acid sequence of SEQ ID NO: 641, and/or (b) a light chainhaving the amino acid sequence of SEQ ID NO: 661.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 684; a CDR2 sequence consisting of SEQ ID NO:686; and a CDR3 sequence consisting of SEQ ID NO: 688; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:704; a CDR2 sequence consisting of SEQ ID NO: 706; and a CDR3 sequenceconsisting of SEQ ID NO: 708. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprisea variable heavy chain comprising an amino acid sequence with at least80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 682,and/or a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:702. In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 682, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 702.In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments may comprise (a) a heavy chain having theamino acid sequence of SEQ ID NO: 681, and/or (b) a light chain havingthe amino acid sequence of SEQ ID NO: 701.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 724; a CDR2 sequence consisting of SEQ ID NO:726; and a CDR3 sequence consisting of SEQ ID NO: 728; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:744; a CDR2 sequence consisting of SEQ ID NO: 746; and a CDR3 sequenceconsisting of SEQ ID NO: 748. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprisea variable heavy chain comprising an amino acid sequence with at least80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 722,and/or a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:742. In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 722, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 742.In a further specific embodiment, the selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a heavy chain havingthe amino acid sequence of SEQ ID NO: 721, and/or (b) a light chainhaving the amino acid sequence of SEQ ID NO: 741.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 764; a CDR2 sequence consisting of SEQ ID NO:766; and a CDR3 sequence consisting of SEQ ID NO: 768; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:784; a CDR2 sequence consisting of SEQ ID NO: 786; and a CDR3 sequenceconsisting of SEQ ID NO: 788. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprisea variable heavy chain comprising an amino acid sequence with at least80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 762,and/or a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:782. In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 762, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 782.In another specific embodiment, selected anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a heavy chain havingthe amino acid sequence of SEQ ID NO: 761, and/or (b) a light chainhaving the amino acid sequence of SEQ ID NO: 781.

The invention also embraces any of the methods disclosed herein whereinanti-PACAP antibodies and antigen binding fragments thereof identifiedby the disclosed methods, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 804; a CDR2 sequence consisting of SEQ ID NO:806; and a CDR3 sequence consisting of SEQ ID NO: 808; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:824; a CDR2 sequence consisting of SEQ ID NO: 826; and a CDR3 sequenceconsisting of SEQ ID NO: 828. In a further specific embodiment, selectedanti-PACAP antibodies and antigen binding fragments thereof may comprisea variable heavy chain comprising an amino acid sequence with at least80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 802,and/or a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:822. In a further embodiment, selected anti-PACAP antibodies and antigenbinding fragments thereof may comprise (a) a variable heavy chain havingthe amino acid sequence of SEQ ID NO: 802, and/or (b) a variable lightchain having the amino acid sequence of SEQ ID NO: 822. In anotherspecific embodiment, selected anti-PACAP antibodies and antigen bindingfragments thereof may comprise (a) a heavy chain having the amino acidsequence of SEQ ID NO: 801, and/or (b) a light chain having the aminoacid sequence of SEQ ID NO: 821.

Additionally, another aspect of the invention generally relates to anyof the methods disclosed herein wherein the anti-PACAP antibody orfragment thereof may comprise: a light chain and/or a heavy chain, whichhas a framework (FR) region and one or more Complementary DeterminingRegion(s) (CDR), and wherein one or more of the FR or CDR residues inthe light chain and/or heavy chain may be substituted with another aminoacid residue. Also, the invention pertains to any of the methodsdisclosed herein wherein the antibody or fragment thereof may be ahumanized antibody or fragment thereof; and/or wherein the antibody orfragment thereof may be a chimeric antibody or fragment thereof; and/orwherein the antibody or fragment thereof may comprise a single chainantibody or fragment thereof. Said chimeric antibody or fragment thereofaccording to the invention may comprise a human Fc, e.g., wherein thehuman Fc may be derived from IgG1, IgG2, IgG3, or IgG4.

Furthermore, the invention generally pertains to any of the methodsdisclosed herein wherein anti-PACAP antibodies or fragments thereof maycomprise a polypeptide sequence that may have at least 90% or greaterhomology to two of the polypeptide sequences of the invention.Additionally, the invention relates to any of the methods disclosedherein wherein the anti-PACAP antibody or fragment thereof may comprisea polypeptide sequence having at least 95% or greater homology to anyone of the polypeptide sequences of the invention.

Moreover, another aspect of the invention embraces any of the methodsdisclosed herein wherein the anti-PACAP antibody or fragment thereof maybind to PACAP with a binding affinity (K_(D)) of less than or equal to5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸M,5×10⁻⁹ M, 10⁻⁹M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹² M, 5×10⁻¹³ M, or 10⁻¹³ M, as determined by ELISA, bio-layerinterferometry (“BLI”), KINEXA, or surface plasmon resonance at 25° C.or 37° C. Furthermore, the invention embraces a method wherein theanti-PACAP antibody or fragment thereof may bind to PACAP with a K_(D)that may be less than about 100 nM, less than about 40 nM, less thanabout 1 nM, less than about 100 pM, less than about 50 pM, or less thanabout 25 pM, and/or wherein the anti-PACAP antibody or fragment thereofmay bind to PACAP with a K_(D) that may be between about 10 pM and about100 pM.

Another embodiment of the invention relates to any of the methodsdisclosed herein wherein the antibody or fragment thereof may beentirely non-glycosylated, or may lack N-glycosylation, or may containonly mannose residues. An additional embodiment of the inventionpertains to any of the methods disclosed herein wherein the antibody orfragment thereof may contain an Fc region that may be modified to altereffector function, half-life, proteolysis, and/or glycosylation. Theinvention additionally embraces any of the methods disclosed hereinwherein the anti-PACAP antibody or fragment thereof may specificallybind to circulating soluble PACAP molecules in vivo. An additionalembodiment of the invention encompasses any of the methods disclosedherein wherein the anti-PACAP antibody or fragment thereof mayspecifically bind to PACAP27 and/or PACAP38.

In yet another embodiment, the invention embraces any of the methodsdisclosed herein wherein the affinity of said anti-PACAP antibody orfragment thereof for PACAP may be at least 10-fold, 30-fold, 100-fold,300-fold, 1000-fold, 3000-fold, 10000-fold, 30000-fold, 100000-fold,300000-fold, 1000000-fold, 3000000-fold, 10000000-fold, 30000000-fold ormore stronger than the affinity of said anti-PACAP antibody and antigenbinding fragment to VIP. Also, the invention pertains to any of themethods disclosed herein wherein the anti-PACAP antibody or fragmentthereof may (a) inhibit or neutralize at least one biological effectelicited by PACAP; (b) neutralize or inhibit PACAP activation of atleast one of PAC1-R, VPAC1-R and/or VPAC2-R; (c) neutralize or inhibitPACAP activation of each of PAC1-R, VPAC1-R and VPAC2-R; (d) neutralizeor inhibit PACAP activation of PAC1-R; (e) inhibits PACAP binding to atleast one of PAC1-R, VPAC1-R and/or VPAC2-R; (f) inhibits PACAP bindingto each of PAC1-R, VPAC1-R and/or VPAC2-R; (g) inhibits PACAP binding toPAC1-R; and/or (h) inhibits PACAP-induced cAMP production. Additionally,the invention encompasses any of the methods disclosed herein whereinthe anti-PACAP antibody or fragment thereof may inhibit the associationof PACAP with one or more PACAP receptors including PAC-1R, VPAC1-R,and/or VPAC2-R.

An additional embodiment of the invention embraces any of the methodsdisclosed herein wherein the antibody or fragment thereof may beadministered intramuscularly, subcutaneously, intravenously, rectally,by infusion, orally, transdermally, or by inhalation. Also, theinvention relates to any of the methods disclosed herein wherein theantibody or fragment thereof may be administered intravenously. Also,the invention encompasses any of the methods disclosed herein whereinthe antibody or fragment thereof may be administered with an additionaltherapeutic agent or regimen selected from anti-histamines,anti-inflammatory agents, and antibiotics. Also, another embodiment ofthe invention pertains to any of the methods disclosed herein whereinthe antibody or fragment thereof may be directly or indirectly attachedto a detectable label or therapeutic agent. The invention furtherrelates to any of the methods disclosed herein wherein the antibody orfragment thereof may further comprise an effector moiety. Said effectormoiety may be a detectable moiety or a functional moiety, e.g., whereinsaid detectable moiety may be a fluorescent dye, an enzyme, a substrate,a bioluminescent material, a radioactive material, or a chemiluminescentmaterial, or said functional moiety may be streptavidin, avidin, biotin,a cytotoxin, a cytotoxic agent, or a radioactive material.

Another aspect of the invention generally relates to a method ofassessing the potential in vivo efficacy of a candidate anti-PituitaryAdenylate Cyclase-Activating Peptide (PACAP) antibody, an anti-PituitaryAdenylate Cyclase Activating Polypeptide type 1 Receptor (PAC1-R)antibody, and antigen binding fragment thereof for treatingPACAP-associated photophobia or light aversion, that may comprisedetermining whether the antibody or fragment thereof may inhibit ordiminish light aversion behavior in a rodent administered PACAP, ascompared to a rodent administered PACAP in the presence of one or moreof the candidate anti-PACAP, anti-PAC1-R antibody or fragment thereof.For example, said method of assessment may be used to assess whether theantibody or fragment thereof may be effective for treatment of aneurological condition characterized by increased PACAP levels, and/orsaid method of assessment may be used to assess whether the antibody orfragment thereof may be effective for treatment of migraine, menstrualmigraine, or chronic migraine. Additionally, said method of assessmentmay be used to assess whether the antibody or fragment thereof may beeffective for treatment of migraines (with or without aura), weightloss, cancer or tumors, angiogenesis associated with cancer or tumorgrowth, angiogenesis associated with cancer or tumor survival,hemiplegic migraines, cluster headaches, migrainous neuralgia, chronicheadaches, tension headaches, general headaches, headache-free migraine,abdominal migraine, hot flashes, chronic paroxysomal hemicrania,secondary headaches caused by an underlying structural problem in thehead or neck, cranial neuralgia, sinus headaches, allergy-inducedheadaches or allergy-induced migraines, pain, inflammatory pain,post-operative incision pain, complex regional pain syndrome, cancerpain, primary or metastatic bone cancer pain, fracture pain,osteoporotic fracture pain, pain resulting from burn, osteoporosis, goutjoint pain, pain associated with sickle cell crises, nociceptive pain,hepatocellular carcinoma, breast cancer, liver cirrhosis, neurogenicpain, neuropathic pain, trigeminal neuralgia, post-herpetic neuralgia,phantom limb pain, fibromyalgia, menstrual pain, ovarialgia, reflexsympathetic dystrophy, neurogenic pain, osteoarthritis or rheumatoidarthritis pain, lower back pain, diabetic neuropathy, sciatica, visceralpain associated with gastro-esophageal reflux, dyspepsia, irritablebowel syndrome, inflammatory bowel disease, Crohn's disease, ileitis,ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstrualperiod, labor, menopause, prostatitis, or pancreatitis.

An additional embodiment of the invention relates to any of the methodsdisclosed herein wherein any of said methods may further compriseadministering an additional active agent selected from a beta-blocker,flunarizine, valproic acid, topiramate, amitriptyline, venlafaxine,gabapentin, naproxen, butterbur root, vitamin B2, and/or magnesium.Also, said additional active agent may be selected from agonists,antagonists, and modulators of TNF-α, IL-2, IL-4, IL-6, IL-10, IL-12,IL-13, IL-18, IFN-α, IFN-γ, BAFF, CXCL13, IP-10, VEGF, EPO, EGF, HRG,Hepatocyte growth Factor (HGF), hepcidin, antibodies reactive againstany of the foregoing, and antibodies reactive against any of theirreceptors. Yet another embodiment of the invention embraces any of themethods disclosed herein wherein any of said methods may furthercomprise administering analgesics, anti-histamines, antipyretics,anti-inflammatories, antibiotics, antivirals, and/or anti-cytokineagents.

An additional embodiment of the invention relates to any of the methodsdisclosed herein wherein any of said methods may further compriseadministering an active agent that may be selected from one or more ofthe following: 2-arylpropionic acids, aceclofenac, acemetacin,acetylsalicylic acid, alclofenac, alminoprofen, amoxiprin, ampyrone,arylalkanoic acids, azapropazone, benorylate/benorilate, benoxaprofen,bromfenac, carprofen, celecoxib, choline magnesium salicylate,clofezone, COX-2 inhibitors, dexibuprofen, dexketoprofen, diclofenac,diflunisal, droxicam, ethenzamide, etodolac, etoricoxib, faislamine,fenamic acids, fenbufen, fenoprofen, flufenamic acid, flunoxaprofen,flurbiprofen, ibuprofen, ibuproxam, indomethacin, indoprofen, kebuzone,ketoprofen, ketorolac, lornoxicam, loxoprofen, lumiracoxib, magnesiumsalicylate, meclofenamic acid, mefenamic acid, meloxicam, metamizole,methyl salicylate, mofebutazone, nabumetone, naproxen, N-arylanthranilicacids, Nerve Growth Factor (NGF), oxametacin, oxaprozin, oxicams,oxyphenbutazone, oxytocin, parecoxib, phenazone, phenylbutazone,phenylbutazone, piroxicam, pirprofen, profens, proglumetacin,pyrazolidine derivatives, rofecoxib, salicyl salicylate, salicylamide,salicylates, substance P, sulfinpyrazone, sulindac, suprofen, tenoxicam,tiaprofenic acid, tolfenamic acid, tolmetin, and valdecoxib.

Additionally, the invention relates to any of the methods disclosedherein wherein any of said methods may further comprise administering ananti-histamine active agent that may be selected from one or more of thefollowing: acrivastine, astemizole, azatadine, azelastine, betatastine,brompheniramine, buclizine, cetirizine, cetirizine analogues,chlorpheniramine, clemastine, CS 560, cyproheptadine, desloratadine,dexchlorpheniramine, ebastine, epinastine, fexofenadine, HSR 609,hydroxyzine, levocabastine, loratidine, methscopolamine, mizolastine,norastemizole, phenindamine, promethazine, pyrilamine, terfenadine, andtranilast. Moreover, the invention encompasses any of the methodsdisclosed herein where any of said methods may further compriseadministering an antibiotic active agent that may be selected from oneor more of the following: amikacin, aminoglycosides, amoxicillin,ampicillin, ansamycins, arsphenamine, azithromycin, azlocillin,aztreonam, bacitracin, carbacephem, carbapenems, carbenicillin,cefaclor, cefadroxil, cefalexin, cefalothin, cefalotin, cefamandole,cefazolin, cefdinir, cefditoren, cefepime, cefixime, cefoperazone,cefotaxime, cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten,ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cephalosporins,chloramphenicol, cilastatin, ciprofloxacin, clarithromycin, clindamycin,cloxacillin, colistin, co-trimoxazole, dalfopristin, demeclocycline,dicloxacillin, dirithromycin, doripenem, doxycycline, enoxacin,ertapenem, erythromycin, ethambutol, flucloxacillin, fosfomycin,furazolidone, fusidic acid, gatifloxacin, geldanamycin, gentamicin,glycopeptides, herbimycin, imipenem, isoniazid, kanamycin, levofloxacin,lincomycin, linezolid, lomefloxacin, loracarbef, macrolides, mafenide,meropenem, methicillin, metronidazole, mezlocillin, minocycline,monobactams, moxifloxacin, mupirocin, nafcillin, neomycin, netilmicin,nitrofurantoin, norfloxacin, ofloxacin, oxacillin, oxytetracycline,paromomycin, penicillin, penicillins, piperacillin, platensimycin,polymyxin B, polypeptides, prontosil, pyrazinamide, quinolones,quinupristin, rifampicin, rifampin, roxithromycin, spectinomycin,streptomycin, sulfacetamide, sulfamethizole, sulfanilamide,sulfasalazine, sulfisoxazole, sulfonamides, teicoplanin, telithromycin,tetracycline, tetracyclines, ticarcillin, tinidazole, tobramycin,trimethoprim, trimethoprim-sulfamethoxazole, troleandomycin,trovafloxacin, and vancomycin.

Also, the invention relates to any of the methods disclosed hereinwherein any of said methods may further comprise administering an activeagent that may be selected from: aldosterone, beclomethasone,betamethasone, corticosteroids, cortisol, cortisone acetate,deoxycorticosterone acetate, dexamethasone, fludrocortisone acetate,glucocorticoids, hydrocortisone, methylprednisolone, prednisolone,prednisone, steroids, and triamcinolone, and combinations thereof. Anadditional embodiment of the invention pertains to any of the methodsdisclosed herein wherein any of said methods may further compriseadministering an active agent that ma be selected from: ibuprofen,naproxen, sumatriptan, paracetamol/acetaminophen, caffeine, a triptan, acorticosteroid, an anti-mimetic, and combinations thereof.

Another aspect of the invention generally encompasses a method ofscreening for antibodies or antigen binding fragments that may decreaseor inhibit light-induced pain signaling through intrinsicallyphotosensitive retinal ganglion cells (“ipRGCs”), that may comprise:

(i) providing at least one first test subject and at least one secondtest subject;

(ii) administering PACAP to the first test subject and the second testsubject;

(iii) administering to the first test subject one or more ofanti-Pituitary Adenylate Cyclase-Activating Peptide (PACAP) antibodies,anti-Pituitary Adenylate Cyclase Activating Polypeptide type 1 Receptor(PAC1-R) antibodies, anti-vasoactive intestinal peptide receptor type 1(VPAC1-R) antibodies, or anti-vasoactive intestinal peptide receptortype 2 (VPAC2-R) antibodies, preferably an anti-PACAP antibody oranti-PAC1-R antibody, or an antigen binding fragment of any of theforegoing;(iv) comparing the response of the at least one first test subject andat least one second test subject to light; and(v) based on this comparison identifying one or more antibodies thatdecrease or inhibit light-induced pain signaling through intrinsicallyphotosensitive retinal ganglion cells (“ipRGCs”).

An additional embodiment of the invention relates to a method ofscreening for a test substance that may treat or prevent photophobia orlight aversion in a subject in need thereof by decreasing or inhibitinglight-induced pain signaling through intrinsically photosensitiveretinal ganglion cells (“ipRGCs”), that may comprise:

(i) providing at least one first test subject and at least one secondtest subject;

(ii) administering PACAP to the first test subject and the second testsubject;

(iii) administering to the first test subject one or more ofanti-Pituitary Adenylate Cyclase-Activating Peptide (PACAP) antibodies,anti-Pituitary Adenylate Cyclase Activating Polypeptide type 1 Receptor(PAC1-R) antibodies, anti-vasoactive intestinal peptide receptor type 1(VPAC1-R) antibodies, or anti-vasoactive intestinal peptide receptortype 2 (VPAC2-R) antibodies, preferably an anti-PACAP antibody oranti-PAC1-R antibody, or an antigen binding fragment of any of theforegoing;(iv) comparing the response of the at least one first test subject andat least one second test subject to light; and(v) based on this comparison, identifying one or more antibodies orantigen binding fragments thereof that may be useful for treating orpreventing light-induced pain signaling through ipRGCs.

An aspect of the present invention in general relates to anti-PACAPantibodies and antigen binding fragments thereof, preferably human,humanized, or chimerized anti-human PACAP antibodies or antibodyfragments thereof that may antagonize, inhibit, neutralize or block atleast one biological effect associated with human PACAP.

Moreover, the invention pertains to anti-PACAP antibodies and antigenbinding fragments thereof that may include human, humanized orchimerized anti-PACAP antibodies or antibody fragments thereof thatspecifically compete for binding to human PACAP with an antibodyselected from the group consisting of Ab1, Ab2, Ab13, Ab14, Ab15, Ab16,Ab17, Ab18, Ab19, Ab1.H, Ab5, Ab7, Ab11, Ab12, Ab4, Ab3, Ab6, Ab8, andAb9, or an antigen-binding fragment thereof.

Additionally, the anti-PACAP antibodies and antigen binding fragments ofthe invention may include human, humanized or chimerized anti-PACAPantibodies or antibody fragments that may specifically bind to at leastone linear or conformational epitope bound by an anti-PACAP antibodyselected from the group consisting of Ab1, Ab2, Ab13, Ab14, Ab15, Ab16,Ab17, Ab18, Ab19, Ab1.H, Ab5, Ab7, Ab11, Ab12, Ab4, Ab3, Ab6, Ab8, andAb9 or an antigen-binding fragment thereof. The epitope may beidentified by alanine scanning, e.g., as disclosed in Example 12, oranother art-recognized method. Also, in another embodiment, theanti-PACAP antibodies and antigen binding fragments thereof of theinvention may include human, humanized or chimerized anti-PACAPantibodies or antibody fragments thereof which may bind to the identicalepitopes as any one of Ab1, Ab2, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18,Ab19, Ab1.H, Ab5, Ab7, Ab11, Ab12, Ab4, Ab3, Ab6, Ab8, and Ab9 or anantigen-binding fragment thereof. The epitope may be identified byalanine scanning, e.g., as disclosed in Example 12, or anotherart-recognized method.

In a further embodiment of the invention, the anti-PACAP antibodies orantigen binding fragments thereof of the invention may include human,humanized or chimerized anti-PACAP antibodies or antibody fragmentswhich may specifically bind to an epitope on human PACAP or a fragmentor variant thereof containing the corresponding amino acid residueswherein said epitope includes one or more of the following:

-   -   i. at least one of residues 7, 10, 13, and 14 of a human PACAP;    -   ii. at least one of residues 5, 6, 8, 10, and 13 of a human        PACAP;    -   iii. at least one of residues 6, 8, 9, 10, and 13 of a human        PACAP;    -   iv. at least one of residues 5, 6, 8, 9, 10, and 13 of a human        PACAP;    -   v. at least one of residues 7, 10, 12, 13, 14, and 17 of a human        PACAP;    -   vi. at least one of residues 5, 6, 8, 9, 10, 12, and 13 of a        human PACAP;    -   vii. at least one of residues 5, 6, 8, 9, 10, 13, and 14 of a        human PACAP;    -   viii. at least one of residues 6, 8, 10, 11, 13, 14, and 18 of a        human PACAP;    -   ix. at least one of residues 8, 9, 10, 13, 14, 17, and 18 of a        human PACAP;    -   x. at least one of residues 3, 4, 5, 6, 7, 10, 13, and 14 of a        human PACAP;    -   xi. at least one of residues 5, 6, 8, 9, 10, 12, 13, and 14 of a        human PACAP;    -   xii. at least one of residues 5, 6, 9, 10, 12, 13, 14, and 17 of        a human PACAP;    -   xiii. at least one of residues 6, 8, 10, 11, 13, 14, 18, and 22        of a human PACAP;    -   xiv. at least one of residues 8, 9, 10, 11, 12, 13, 14, 17, and        21 of a human PACAP;    -   xv. at least one of residues 4, 5, 6, 8, 9, 10, 12, 13, 14, and        17 of a human PACAP;    -   xvi. at least two of the residues of any one of (i)-(xv);    -   xvii. at least three of the residues of any one of (i)-(xv);    -   xviii. at least four of the residues of any one of (i)-(xv);    -   xix. at least five of the residues of any one of (ii)-(xv);    -   xx. at least six of the residues of any one of (iv)-(xv);    -   xxi. at least seven of the residues of any one of (vi)-(xv);    -   xxii. at least eight of the residues of any one of (x)-(xv);    -   xxiii. at least nine of the residues of (xiv) or (xv); and    -   xxiv. all ten residues of (xv);

In a specific embodiment of the invention, anti-PACAP antibodies orantigen binding fragments thereof of the invention may include human,humanized or chimerized anti-PACAP antibodies or antibody fragmentswhich specifically bind to an epitope on human PACAP, or a fragment orvariant thereof that may contain the corresponding amino acid residuesthat may include residues 8 and/or 14 of human PACAP. Also, in anotherembodiment of the invention, the anti-PACAP antibodies or antigenbinding fragments thereof may include a human, humanized or chimerizedanti-PACAP antibodies or antibody fragments thereof, which specificallybind to an epitope on human PACAP (or a fragment or variant thereofcontaining the corresponding amino acid residues that may be present inhuman wild-type PACAP38) but not human wild-type human PACAP27. Theepitope may be identified by alanine scanning, e.g., as disclosed inExample 12 or another art-recognized method.

In an additional embodiment of the invention, anti-PACAP antibodies orantigen binding fragments thereof of the invention may include human,humanized or chimerized anti-PACAP antibodies or antibody fragmentsthereof which may specifically bind to an epitope on human PACAP or afragment or variant thereof that contain the corresponding amino acidresidues, wherein said epitope may consist of the residues of any one of(i)-(xv) as described above. The epitope may be identified by alaninescanning, e.g., as disclosed in Example 12, or another art-recognizedmethod.

Another aspect of the invention also embraces anti-PACAP antibodies orantigen binding fragments thereof that may include human, humanized orchimerized anti-PACAP antibodies or antibody fragment which specificallybind to an epitope on human PACAP (or a fragment or variant thereof thatmay contain the corresponding amino acid residues) that may be presentin human wild-type PACAP38 and in human wild-type human PACAP27.

Additionally, the anti-PACAP antibodies or antigen binding fragmentsthereof may include human, humanized or chimerized anti-PACAP antibodiesor antibody fragments thereof, which may specifically bind to humanwild-type human PACAP38 but which may not bind or appreciably bind tohuman wild-type human PACAP27. The invention may also embody anti-PACAPantibodies or antigen binding fragments thereof that may include human,humanized or chimerized anti-PACAP antibodies or antibody fragmentsthereof which may specifically interact with residues 28 and 31 of humanPACAP38. In another embodiment of the invention, the anti-PACAPantibodies and antigen binding fragments thereof of the invention mayinclude human, humanized or chimerized anti-PACAP antibodies or antibodyfragments thereof which may have a K_(D) for human PACAP38 which may beat least 10, 100, 1000, 10,000 or 100,000 fold lower (stronger) than theK_(D) of said antibody or antibody fragment to human PACAP27.

In another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof of the invention may include human, humanized orchimerized anti-PACAP antibodies or antibody fragments thereof which donot bind to or do not appreciably bind to human Vasoactive IntestinalPeptide (“VIP”). Said anti-PACAP antibodies or antibody fragmentsthereof may have a KD for human PACAP which may be at least 10, 100,1000, 10,000 or 100,000 fold lower (stronger) than the KD of saidantibody or antibody fragment to human VIP.

In embodiments of the invention, a human, humanized or chimerizedanti-PACAP antibody or antibody fragment as disclosed herein, mayinhibit or may neutralize at least one biological effect elicited byhuman PACAP.

In another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof of the invention may include human, humanized orchimerized anti-PACAP antibodies or antibody fragments thereof that maycomprise one or more of the following properties: (a) inhibit, block orprevent PACAP activation of at least one of PAC1 receptor (“PAC1-R”),vasoactive intestinal peptide receptor type 1 (“VPAC1-R”), and/orvasoactive intestinal peptide receptor type 2 (“VPAC2-R”); (b) inhibit,block or prevent PACAP activation of each of PAC1-R, VPAC1-R, andVPAC2-R; (c) inhibit, block or prevent PACAP activation of PAC1-R; (d)are capable of inhibiting PACAP binding to at least one of PAC1-R,VPAC1-R, and/or VPAC2-R; (e) are capable of inhibiting PACAP binding toeach of PAC1-R, VPAC1-R, and/or VPAC2-R; (f) may be capable ofinhibiting PACAP binding to PAC1-R-expressing cells; (g) are capable ofinhibiting PACAP binding to VPAC1-R-expressing cells; (h) are capable ofinhibiting PACAP binding to VPAC2-R-expressing cells; (i) do inhibitPACAP binding to the cell surface, e.g. via a glycosaminoglycan (“GAG”);(j) do not inhibit PACAP-mediated binding of such antibody to the cellsurface, e.g., via a GAG; (k) do inhibit PACAP-mediated binding of suchantibody to the cell surface, e.g., via a glycosaminoglycan (“GAG”); (l)inhibit, block or prevent PACAP-induced cAMP production; and/or (m) whenadministered to a subject reduce PACAP-induced vasodilation,photophobia, mast cell degranulation and/or neuronal activation.

The invention may also pertain to anti-PACAP antibodies and antigenbinding fragments thereof that may be preferably human, humanized orchimerized anti-PACAP antibodies or antibody fragments which may besubstantially non-immunogenic in human subjects. The invention may alsorelate to anti-PACAP antibodies and antigen binding fragments thereofthat may be preferably human, humanized or chimerized anti-PACAPantibodies or antibody fragments, which may be suitable for treating ahuman subject having an acute, episodic or chronic condition associatedwith increased vasodilation, photophobia, mast cell degranulation and/orneuronal activation.

The invention may also embody anti-PACAP antibodies and antigen bindingfragments thereof that may be preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments that may comprise at least 2complementarity determining regions (“CDRs”) of an anti-PACAP antibodythat may be selected from Ab1, Ab2, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18,Ab19, Ab1.H, Ab5, Ab7, Ab11, Ab12, Ab4, Ab3, Ab6, Ab8, or Ab9,preferably including the V_(H) CDR3 and/or the V_(L) CDR3. Anotheradditional embodiment of the invention may include anti-PACAP antibodiesand antigen binding fragments thereof that may be preferably human,humanized or chimerized anti-PACAP antibodies or antibody fragments thatmay comprise at least 3, at least 4, at least 5, or all 6 CDRS of ananti-PACAP antibody selected from Ab1, Ab2, Ab13, Ab14, Ab15, Ab16,Ab17, Ab18, Ab19, Ab1.H, Ab5, Ab7, Ab11, Ab12, Ab4, Ab3, Ab6, Ab8, orAb9. In instances where all 6 CDRs may not be present, preferably atleast the V_(H) CDR3 and the V_(L) CDR3 may be present.

In another embodiment of the invention, anti-PACAP antibodies andantigen binding fragments thereof, preferably human, humanized orchimerized anti-PACAP antibodies or antibody fragments thereof, maycomprise a sequence variant of any of the antibodies or antibodyfragments of the invention that may contain one or more modificationsthat may putatively alter binding affinity or immunogenicity.

In a specific embodiment, anti-PACAP antibodies and antigen bindingfragments thereof according to the invention, comprise human, humanized,or chimerized anti-PACAP antibodies or antigen binding fragmentsthereof, which comprise (a) a variable heavy chain comprising a CDR1sequence consisting of SEQ ID NO: 4; a CDR2 sequence consisting of SEQID NO: 6; and a CDR3 sequence consisting of SEQ ID NO: 8; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:24; a CDR2 sequence consisting of SEQ ID NO: 26; and a CDR3 sequenceconsisting of SEQ ID NO: 28. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof may comprise (a) a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 2, and/or (b) avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 22. In amore specific embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof may comprise (a) a variable heavy chain having theamino acid sequence of SEQ ID NO: 2, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 22. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 1, and/or(b) a light chain having the amino acid sequence of SEQ ID NO: 21.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, preferably human,humanized, or chimerized anti-PACAP antibodies and antigen bindingfragments thereof, comprise (a) a variable heavy chain comprising a CDR1sequence consisting of SEQ ID NO: 44; a CDR2 sequence consisting of SEQID NO: 46; and a CDR3 sequence consisting of SEQ ID NO: 48; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:64; a CDR2 sequence consisting of SEQ ID NO: 66; and a CDR3 sequenceconsisting of SEQ ID NO: 68. More specifically, the anti-PACAPantibodies and antigen binding fragments thereof comprise (a) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 42, and/or (b) avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 62. Inanother specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof comprise (a) a variable heavy chain having theamino acid sequence of SEQ ID NO: 42, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 62. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof may comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 41,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:61.

In another specific embodiment, anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, preferably human,humanized, or chimerized anti-PACAP antibodies and antigen bindingfragments thereof, may comprise (a) a variable heavy chain comprising aCDR1 sequence consisting of SEQ ID NO: 84; a CDR2 sequence consisting ofSEQ ID NO: 86; and a CDR3 sequence consisting of SEQ ID NO: 88; and/or(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO: 104; a CDR2 sequence consisting of SEQ ID NO: 106; and a CDR3sequence consisting of SEQ ID NO: 108. Alternatively, the anti-PACAPantibodies and antigen binding fragments thereof can comprise (a) avariable heavy chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 82,and/or (b) a variable light chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 102. In another specific embodiment, the anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 82, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 102.More specifically, the anti-PACAP antibodies and antigen bindingfragments thereof can comprise (a) a heavy chain having the amino acidsequence of SEQ ID NO: 81, and/or (b) a light chain having the aminoacid sequence of SEQ ID NO: 101.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, preferably human,humanized, or chimerized anti-PACAP antibodies and antigen bindingfragments thereof, may comprise (a) a variable heavy chain comprising aCDR1 sequence consisting of SEQ ID NO: 124; a CDR2 sequence consistingof SEQ ID NO: 126; and a CDR3 sequence consisting of SEQ ID NO: 128;and/or (b) a variable light chain comprising a CDR1 sequence consistingof SEQ ID NO: 144; a CDR2 sequence consisting of SEQ ID NO: 146; and aCDR3 sequence consisting of SEQ ID NO: 148. Alternatively, theanti-PACAP antibodies and antigen binding fragments thereof may comprise(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:122 and/or (b) a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 142. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof may comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 122, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 142.More specifically, the anti-PACAP antibodies and antigen bindingfragments thereof can comprise (a) a heavy chain having the amino acidsequence of SEQ ID NO: 121, and/or (b) a light chain having the aminoacid sequence of SEQ ID NO: 141.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, preferably human,humanized, or chimerized anti-PACAP antibodies and antigen bindingfragments thereof, may comprise (a) a variable heavy chain comprising aCDR1 sequence consisting of SEQ ID NO: 164; a CDR2 sequence consistingof SEQ ID NO: 166; and a CDR3 sequence consisting of SEQ ID NO: 168;and/or (b) a variable light chain comprising a CDR1 sequence consistingof SEQ ID NO: 184; a CDR2 sequence consisting of SEQ ID NO: 186; and aCDR3 sequence consisting of SEQ ID NO: 188. Alternatively, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:162, and/or (b) a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 182. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof comprise (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 162, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO: 182. Morespecifically, the anti-PACAP antibodies and antigen binding fragmentsthereof can comprise (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 161, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 181.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 204; a CDR2 sequenceconsisting of SEQ ID NO: 206; and a CDR3 sequence consisting of SEQ IDNO: 208; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 224; a CDR2 sequence consisting of SEQ ID NO:226; and a CDR3 sequence consisting of SEQ ID NO: 228. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof cancomprise (a) a variable heavy chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 202 and/or (b) a variable light chain comprising an aminoacid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO: 222. In another embodiment, the anti-PACAPantibodies and antigen binding fragments thereof comprise (a) a variableheavy chain having the amino acid sequence of SEQ ID NO: 202, and/or (b)a variable light chain having the amino acid sequence of SEQ ID NO: 222.More specifically, the anti-PACAP antibodies and antigen bindingfragments thereof can comprise (a) a heavy chain having the amino acidsequence of SEQ ID NO: 201, and/or (b) a light chain having the aminoacid sequence of SEQ ID NO: 221.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 244; a CDR2 sequenceconsisting of SEQ ID NO: 246; and a CDR3 sequence consisting of SEQ IDNO: 248; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 264; a CDR2 sequence consisting of SEQ ID NO:266; and a CDR3 sequence consisting of SEQ ID NO: 268. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof cancomprise (a) a variable heavy chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 242 and/or (b) a variable light chain comprising an aminoacid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO: 262. In another embodiment, the anti-PACAPantibodies and antigen binding fragments thereof comprise (a) a variableheavy chain having the amino acid sequence of SEQ ID NO: 242, and/or (b)a variable light chain having the amino acid sequence of SEQ ID NO: 262.More specifically, the anti-PACAP antibodies and antigen bindingfragments thereof can comprise (a) a heavy chain having the amino acidsequence of SEQ ID NO: 241, and/or (b) a light chain having the aminoacid sequence of SEQ ID NO: 261.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 284; a CDR2 sequenceconsisting of SEQ ID NO: 286; and a CDR3 sequence consisting of SEQ IDNO: 288; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 304; a CDR2 sequence consisting of SEQ ID NO:306; and a CDR3 sequence consisting of SEQ ID NO: 308. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof cancomprise a variable heavy chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 282, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 302. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof comprise (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 282, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO: 302. Morespecifically, the anti-PACAP antibodies and antigen binding fragmentsthereof can comprise (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 281, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 301.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 324; a CDR2 sequenceconsisting of SEQ ID NO: 326; and a CDR3 sequence consisting of SEQ IDNO: 328; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 344; a CDR2 sequence consisting of SEQ ID NO:346; and a CDR3 sequence consisting of SEQ ID NO: 348. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof cancomprise a variable heavy chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 322, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 342. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof comprise (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 322, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO: 342. Morespecifically, the anti-PACAP antibodies and antigen binding fragmentsthereof can comprise (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 321, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 341.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 364; a CDR2 sequenceconsisting of SEQ ID NO: 366; and a CDR3 sequence consisting of SEQ IDNO: 368; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 384; a CDR2 sequence consisting of SEQ ID NO:386; and a CDR3 sequence consisting of SEQ ID NO: 388. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof cancomprise a variable heavy chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 362, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 382. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof comprise (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 362, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO: 382. Morespecifically, the anti-PACAP antibodies and antigen binding fragmentsthereof can comprise (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 361, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 381.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 484; a CDR2 sequenceconsisting of SEQ ID NO: 486; and a CDR3 sequence consisting of SEQ IDNO: 488; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 504; a CDR2 sequence consisting of SEQ ID NO:506; and a CDR3 sequence consisting of SEQ ID NO: 508. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof cancomprise a variable heavy chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 482, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 502. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof comprise (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 482, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO: 502. Morespecifically, the anti-PACAP antibodies and antigen binding fragmentsthereof can comprise (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 481, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 501.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 524; a CDR2 sequenceconsisting of SEQ ID NO: 526; and a CDR3 sequence consisting of SEQ IDNO: 528; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 544; a CDR2 sequence consisting of SEQ ID NO:546; and a CDR3 sequence consisting of SEQ ID NO: 548. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof cancomprise a variable heavy chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 522, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 542. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof comprise (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 522, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO: 542. Morespecifically, the anti-PACAP antibodies and antigen binding fragmentsthereof can comprise (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 521, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 541.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 564; a CDR2 sequenceconsisting of SEQ ID NO: 566; and a CDR3 sequence consisting of SEQ IDNO: 568; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 584; a CDR2 sequence consisting of SEQ ID NO:586; and a CDR3 sequence consisting of SEQ ID NO: 588. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof cancomprise a variable heavy chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 562, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 582. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof comprise (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 562, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO: 582. Morespecifically, the anti-PACAP antibodies and antigen binding fragmentsthereof can comprise (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 561, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 581.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 604; a CDR2 sequenceconsisting of SEQ ID NO: 606; and a CDR3 sequence consisting of SEQ IDNO: 608; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 624; a CDR2 sequence consisting of SEQ ID NO:626; and a CDR3 sequence consisting of SEQ ID NO: 628. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof cancomprise a variable heavy chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 602, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 622. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof comprise (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 602, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO: 622. Morespecifically, the anti-PACAP antibodies and antigen binding fragmentsthereof can comprise (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 601, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 621.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 644; a CDR2 sequenceconsisting of SEQ ID NO: 646; and a CDR3 sequence consisting of SEQ IDNO: 648; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 664; a CDR2 sequence consisting of SEQ ID NO:666; and a CDR3 sequence consisting of SEQ ID NO: 668. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof cancomprise a variable heavy chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 642, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 662. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof comprise (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 642, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO: 662. Morespecifically, the anti-PACAP antibodies and antigen binding fragmentsthereof can comprise (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 641, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 661.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 684; a CDR2 sequenceconsisting of SEQ ID NO: 686; and a CDR3 sequence consisting of SEQ IDNO: 688; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 704; a CDR2 sequence consisting of SEQ ID NO:706; and a CDR3 sequence consisting of SEQ ID NO: 708. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof cancomprise a variable heavy chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 682, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 702. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof comprise (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 682, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO: 702. Morespecifically, the anti-PACAP antibodies and antigen binding fragmentsthereof can comprise (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 681, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 701.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 724; a CDR2 sequenceconsisting of SEQ ID NO: 726; and a CDR3 sequence consisting of SEQ IDNO: 728; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 744; a CDR2 sequence consisting of SEQ ID NO:746; and a CDR3 sequence consisting of SEQ ID NO: 748. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof cancomprise a variable heavy chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 722, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 742. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof comprise (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 722, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO: 742. Morespecifically, the anti-PACAP antibodies and antigen binding fragmentsthereof can comprise (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 721, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 741.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 764; a CDR2 sequenceconsisting of SEQ ID NO: 766; and a CDR3 sequence consisting of SEQ IDNO: 768; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 784; a CDR2 sequence consisting of SEQ ID NO:786; and a CDR3 sequence consisting of SEQ ID NO: 788. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof fragmentcan comprise a variable heavy chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 762, and/or a variable light chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO: 782. In another embodiment, the anti-PACAPantibodies and antigen binding fragments thereof comprise (a) a variableheavy chain having the amino acid sequence of SEQ ID NO: 762, and/or (b)a variable light chain having the amino acid sequence of SEQ ID NO: 782.More specifically, the anti-PACAP antibodies and antigen bindingfragments thereof can comprise (a) a heavy chain having the amino acidsequence of SEQ ID NO: 761, and/or (b) a light chain having the aminoacid sequence of SEQ ID NO: 781.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are preferablyhuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof, and comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO: 804; a CDR2 sequenceconsisting of SEQ ID NO: 806; and a CDR3 sequence consisting of SEQ IDNO: 808; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 824; a CDR2 sequence consisting of SEQ ID NO:826; and a CDR3 sequence consisting of SEQ ID NO: 828. Alternatively,the anti-PACAP antibodies and antigen binding fragments thereof cancomprise a variable heavy chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 802, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 822. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof comprise (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 802, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO: 822. Morespecifically, the anti-PACAP antibodies and antigen binding fragmentsthereof can comprise (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 801, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 821.

Additionally, the anti-PACAP antibodies and antigen binding fragments ofthe invention may include human, humanized or chimerized anti-PACAPantibodies or antibody fragments wherein the antibodies or antibodyfragments may be selected from the group consisting of scFvs,camelbodies, nanobodies, Immunoglobulin New Antigen Receptor (“IgNAR”),fragment antigen binding (“Fab”) fragments, Fab′ fragments, MetMab likeantibodies, monovalent antibody fragments, and F(ab′)₂ fragments. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments of the invention, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, may substantially orentirely lack N-glycosylation and/or O-glycosylation. Also, theinvention embraces an embodiment of the invention wherein the anti-PACAPantibodies and antigen binding fragments of the invention, preferablyhuman, humanized or chimerized anti-PACAP antibodies or antibodyfragments, may comprise a human constant domain, e.g. that of an IgG1,IgG2, IgG3, or IgG4 antibody or fragment thereof.

An additional embodiment of the invention relates to anti-PACAPantibodies and antigen binding fragments, preferably human, humanized orchimerized anti-PACAP antibodies or antibody fragments, wherein saidantibodies or antibody fragments may comprise an Fc region that may havebeen modified to alter at least one of effector function, half-life,proteolysis, or glycosylation, e.g., wherein the Fc region may containone or more mutations that may alter or eliminate N- and/orO-glycosylation.

In yet another embodiment of the invention, anti-PACAP antibodies andantigen binding fragments, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, may bind to PACAP with abinding affinity (KD) of less than or equal to 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, or 10⁻¹³M, e.g., as may determined by ELISA, bio-layer interferometry (“BLI”),KINEXA or surface plasmon resonance at 25° or 37° C. Also, anotherembodiment of the invention pertains to anti-PACAP antibodies andantigen binding fragments, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, wherein said antibodies orantibody fragments may bind to PACAP with a binding affinity (KD) ofless than or equal to 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,or 10⁻¹² M. Additionally, the anti-PACAP antibodies and antigen bindingfragments, preferably human, humanized or chimerized anti-PACAPantibodies or antibody fragments, of the invention may includeanti-PACAP antibodies or antibody fragments which bind to PACAP with anoff-rate (k_(off)) of less than or equal to 5×10⁻⁴ s⁻¹, 10⁻⁴ s⁻¹, 5×10⁻⁵s⁻¹, or 10⁻⁵ s⁻¹.

Another embodiment of the invention relates to anti-PACAP antibodies andantigen binding fragments, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, wherein said antibodies andantibody fragments may be directly or indirectly attached to adetectable label or therapeutic agent. An additional embodiment of theinvention pertains to anti-PACAP antibodies and antigen bindingfragments, preferably human, humanized or chimerized anti-PACAPantibodies or antibody fragments that when administered to a subject mayinhibit or may neutralize at least one biological effect elicited byPACAP. The anti-PACAP antibodies and antigen binding fragments,preferably human, humanized or chimerized anti-PACAP antibodies orantibody fragments, of the invention may neutralize or may inhibit PACAPactivation of at least one of PAC1-R, VPAC1-R, or VPAC2-R; mayneutralize or may inhibit PACAP activation of each of PAC1-R, VPAC1-R,and VPAC2-R; may neutralize or may inhibit PACAP activation of PAC1-R;may be capable of inhibiting or preventing PACAP binding to at least oneof PAC1-R, VPAC1-R, or VPAC2-R; may be capable of inhibiting orpreventing PACAP binding to each of PAC1-R, VPAC1-R, and VPAC2-R; may becapable of inhibiting or preventing PACAP binding to PAC1-R-expressingcells, VPAC1-R-expressing cells, and/or VPAC2-R-expressing cells; mayinhibit or block PACAP-induced cAMP production; may, when administeredto a subject, may reduce PACAP-induced vasodilation, photophobia, mastcell degranulation and/or neuronal activation.

In yet another embodiment of the invention, anti-PACAP antibodies andantigen binding fragments, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, may bind to PACAP with aK_(D) that may be less than about 100 nM; with a K_(D) that may be lessthan about 40 nM; with a K_(D) that may be less than about 100 pM; witha K_(D) that may be less than about 50 pM; with a K_(D) that may be lessthan about 25 pM; or with a K_(D) that may be between about 10 pM andabout 100 pM. The invention also embraces anti-PACAP antibodies andantigen binding fragments, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, that may have strongerbinding affinity for PACAP as compared to VIP and/or that may not bindto VIP, e.g., wherein said antibodies or antibody fragments thereof mayhave an affinity to PACAP that may be at least 10-fold, 30-fold,100-fold, 300-fold, 1000-fold, 3000-fold, 10000-fold, 30000-fold,100000-fold, 300000-fold, 1000000-fold, 3000000-fold, 10000000-fold,30000000-fold or more stronger than the affinity of said antibody orantibody fragment to VIP.

In another embodiment, the invention pertains to anti-PACAP antibodiesand antigen binding fragments, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, that may be attached to atleast one effector moiety, e.g., wherein said effector moiety maycomprise a chemical linker. In another embodiment, the inventionpertains to anti-PACAP antibodies and antigen binding fragments,preferably human, humanized or chimerized anti-PACAP antibodies orantibody fragments, that may be attached to one or more detectablemoieties, e.g., wherein said detectable moieties may comprise afluorescent dye, enzyme, substrate, bioluminescent material, radioactivematerial, chemiluminescent moiety, and/or mixtures thereof. Also, theanti-PACAP antibodies and antigen binding fragments of the invention,preferably human, humanized or chimerized anti-PACAP antibodies orantibody fragments, may be attached to one or more functional moieties.

Another embodiment of the invention relates to anti-idiotypic antibodiesthat may be produced against anti-PACAP antibodies or antibodyfragments, wherein said anti-idiotypic antibodies optionally mayneutralize one or more biological effects of the anti-PACAP antibody towhich it may bind. This embodiment of the invention may also relate to amethod of using said anti-idiotypic antibody to monitor the in vivolevels of said anti-PACAP antibody or antibody fragment in a subject orto neutralize the in vivo effects of said anti-PACAP antibody in asubject.

In yet another embodiment, the invention pertains to a composition thatmay be suitable for therapeutic, prophylactic, or a diagnostic use,whereby the composition may comprise a therapeutically, prophylacticallyor diagnostically effective amount of at least one anti-PACAP antibodyor antibody fragment or anti-idiotypic antibody, e.g., wherein thecomposition may be suitable for administration via injection, topical,oral, inhalation or transdermal; may be suitable for subcutaneous,intravenous, intramuscular, topical, oral, inhalatory, intranasal,intrabuccal, vaginal, anal, transdermal, intraperitoneal, or intrathecaladministration; and/or wherein the composition may be suitable forsubcutaneous intravenous or intramuscular administration. The inventionalso embraces an embodiment of the invention wherein said composition ofat least one anti-PACAP antibody or antibody fragment or anti-idiotypicantibody may be lyophilized; and/or wherein said composition maycomprise a pharmaceutically acceptable diluent, carrier, solubilizer,emulsifier, preservative, or mixture thereof. Said composition of theinvention may further comprise at least one other active agent, e.g.,wherein the other active agent may be selected from the group consistingof a chemotherapeutic, an analgesic, an anti-inflammatory, animmunosuppressant, a cytokine, an antiproliferative, an antiemeticand/or a cytotoxin. Said composition may also be lyophilized,stabilized, and/or may be formulated for administration by injection.

A further embodiment of the invention embraces an isolated nucleic acidsequence or nucleic acid sequences that may encode an anti-PACAPantibody or antibody fragment or anti-idiotypic antibody, and whereinsaid isolated nucleic acid sequence or nucleic acid sequences may becontained within a vector or vectors. Additionally, in an embodiment ofthe invention, a host cell may comprise said isolated nucleic acidsequence or sequences, wherein said host cell may be a mammalian,bacterial, fungal, yeast, avian, amphibian, plant or insect cell; and/orsaid host cell may be a filamentous fungus or a yeast. Wherein said hostcell may be a yeast cell, the yeast may be selected from the followinggenera: Arxiozyma; Ascobotryozyma; Citeromyces; Debaryomyces; Dekkera;Eremothecium; Issatchenkia; Kazachstania; Kluyveromyces; Kodamaea;Lodderomyces; Pachysolen; Pichia; Saccharomyces; Saturnispora;Tetrapisispora; Torulaspora; Williopsis; and Zygosaccharomyces; or theyeast may be genus Pichia. In an embodiment of the invention, the yeasthost cell may be selected from Pichia pastoris, Pichia methanolica andHansenula polymorpha (Pichia angusta).

The invention also relates to a method of expressing an anti-PACAPantibody or antibody fragment that may comprise culturing any of but notlimited to the host cells disclosed herein under conditions that mayprovide for expression of said antibody or antibody fragment.Additionally, the invention pertains to said method wherein the hostcell may be a yeast cell or CHO cell that may stably express and maysecrete said antibody or antibody fragment. For example, said yeast cellmay be a polyploid yeast that may be made by a method that may comprise:(i) introducing at least one expression vector containing one or moreheterologous polynucleotides encoding said antibody operably linked to apromoter and a signal sequence into a haploid yeast cell; (ii) producingby mating or spheroplast fusion a polyploid yeast from said first and/orsecond haploid yeast cell; (iii) selecting polyploid yeast cells thatstably express said antibody; and (iv) producing stable polyploid yeastcultures from said polyploid yeast cells that stably express saidantibody into the culture medium; and said polyploid yeast may be of thegenus Pichia. Additionally in another embodiment, the invention embracesa method of expressing an anti-PACAP antibody or antibody fragment thatmay comprise culturing a host cell wherein said host cell may be amammalian cell, e.g., a CHO cell.

Additionally, the invention pertains to a method that may block,inhibit, block or neutralize one or more biological effects associatedwith PACAP in a subject that may comprise administering to said subjecta therapeutically or prophylactically effective amount of a human,humanized or chimerized anti-PACAP antibody or antibody fragment thatmay antagonize, inhibit, neutralize or block at least one biologicaleffect associated with human PACAP. Another aspect of the inventionrelates to a method that may block, inhibit, or neutralize one or morebiological effects associated with PACAP in a subject that may compriseadministering to a subject a therapeutically or prophylacticallyeffective amount of a human, humanized or chimerized anti-PACAP antibodyor antibody fragment that may antagonize, inhibit, neutralize or blockat least one biological effect associated with human PACAP and that maynot substantially interact with (bind) VIP.

Another aspect of the invention generally relates to a method that mayblock, inhibit, or neutralize one or more biological effects, e.g.,vasomotor effects, associated with PACAP in a subject that may compriseadministering to a subject a therapeutically or prophylacticallyeffective amount of a human, humanized or chimerized anti-PACAP antibodyor antibody fragment that may comprise one or more of the following:inhibits, blocks or neutralizes at least one biological effect elicitedby PACAP; neutralizes or inhibits PACAP activation of at least one ofPAC1-R, VPAC1-R, and/or VPAC2-R; inhibits, blocks or neutralizes PACAPactivation of each of PAC1-R, VPAC1-R, and VPAC2-R; neutralizes orinhibits PACAP activation of PAC1-R; inhibits PACAP binding to at leastone of PAC1-R, VPAC1-R, and/or VPAC2-R; inhibits PACAP binding to eachof PAC1-R, VPAC1-R, and/or VPAC2-R; inhibits PACAP binding toPAC1-R-expressing cells; inhibits PACAP binding to VPAC1-R and/orVPAC2-R-expressing cells; does not inhibit PACAP-mediated binding ofsuch antibody to the cell surface, e.g., via a glycosaminoglycan(“GAG”); inhibits PACAP binding to the cell surface, e.g. via aglycosaminoglycan (“GAG”); inhibits PACAP-induced cAMP production;and/or, when administered to a subject, reduces PACAP-inducedvasodilation, photophobia, mast cell degranulation and/or neuronalactivation.

Another embodiment of the invention relates to a method that may block,inhibit, or neutralize vasodilation, e.g., vasodilation of the duralarteries, which may be associated with or may be elicited by PACAP in asubject that may comprise administering to a subject a therapeuticallyor prophylactically effective amount of a human, humanized or chimerizedanti-PACAP antibody or antibody fragment that may block, inhibit, orneutralize vasodilation associated with, or elicited by PACAP.

Yet another embodiment of the invention pertains to a method that maytreat or prevent the onset, frequency, severity or duration of headacheor migraine, e.g., wherein said headache or migraine may be selectedfrom migraine with aura, migraine without aura, hemiplegic migraine,cluster headache, migrainous neuralgia, chronic headache, chronicmigraine, medication overuse headache, and tension headache, in asubject that may comprise administering to a subject in need thereof aneffective amount of a human, humanized or chimerized anti-human PACAPantibody or antibody fragment that may elicit one or more of thefollowing effects: inhibits or neutralizes at least one biologicaleffect elicited by PACAP; neutralizes or inhibits PACAP activation of atleast one of PAC1-R, VPAC1-R, and/or VPAC2-R; neutralizes or inhibitsPACAP activation of each of PAC1-R, VPAC1-R, and VPAC2-R; neutralizes orinhibits PACAP activation of PAC1-R; inhibits PACAP binding to at leastone of PAC1-R, VPAC1-R, and/or VPAC2-R; inhibits PACAP binding to eachof PAC1-R, VPAC1-R, and/or VPAC2-R; inhibits PACAP binding toPAC1-R-expressing cells; inhibits PACAP binding to VPAC1-R and/orVPAC2-R-expressing cells; does not inhibit PACAP-mediated binding ofsuch antibody to the cell surface, e.g., via a GAG; inhibits PACAPbinding to the cell surface, e.g. via a glycosaminoglycan (“GAG”);inhibits PACAP-induced cyclic adenosine monophosphate (“cAMP”)production; and/or, when administered to a subject, reducesPACAP-induced vasodilation, photophobia, mast cell degranulation and/orneuronal activation.

Another embodiment of the invention relates to a method of treating ahuman subject that may have an acute, episodic or chronic condition thatmay be associated with at least one of increased vasodilation,photophobia, mast cell degranulation and neuronal activation or acombination of said conditions that may comprise administering to asubject in need thereof an effective amount of an antagonistic human,humanized or chimerized anti-human PACAP antibody or antibody fragment.

The invention also pertains to any of the methods disclosed herein thatmay be effected by the administration of a therapeutically orprophylactically effective amount of at least one human, humanized orchimerized anti-PACAP antibody or antibody fragment; and/or wherein saidanti-PACAP antibody may be a human antibody or antibody fragment; and/orwherein said anti-PACAP antibody may be a humanized antibody or antibodyfragment; and/or wherein said anti-PACAP antibody may be a chimericantibody or antibody fragment.

Another embodiment of the invention also relates to a method wherein ananti-PACAP antibody or antibody fragment of the invention may bind toPACAP27 and/or PACAP38 and may block PACAP27 and/or PACAP38 binding toPAC1-R, VPAC1-R, and/or VPAC2-R. Another embodiment of the inventionpertains to a method wherein said anti-PACAP antibody or antibodyfragment may bind to PACAP27 and/or PACAP38 and may block PACAP27 and/orPACAP38 binding to each of PAC1-R, VPAC1-R, and VPAC2-R. Yet anotherembodiment of the invention relates to a method wherein said anti-PACAPantibody or antibody fragment may bind to PACAP27 and/or PACAP38 and mayblock PACAP27 and/or PACAP38 binding to PAC1-R-expressing cells.Additionally, said anti-PACAP antibody or antibody fragment of theinvention may have an affinity to PACAP that may be at least 10-fold,30-fold, 100-fold, 300-fold, 1000-fold, 3000-fold, 10000-fold,30000-fold, 100000-fold, 300000-fold, 1000000-fold, 3000000-fold,10000000-fold, 30000000-fold or more stronger than the affinity of saidantibody or antibody fragment to VIP.

The invention embraces a method that may block, inhibit, block orneutralize one or more biological effects associated with PACAP in asubject that may comprise administering to said subject atherapeutically or prophylactically effective amount of a human,humanized or chimerized anti-PACAP antibody or antibody fragment thatmay antagonize, inhibit, neutralize or blocks at least one biologicaleffect associated with human PACAP, and wherein said subject may have acondition selected from the group consisting of migraine with aura,migraine without aura, hemiplegic migraines, cluster headaches,migrainous neuralgia, chronic headaches, tension headaches, generalheadaches, hot flush, photophobia, chronic paroxysmal hemicrania,secondary headaches due to an underlying structural problem in the head,secondary headaches due to an underlying structural problem in the neck,cranial neuralgia, sinus headaches, headache associated with sinusitis,allergy-induced headaches, allergy-induced migraines, trigeminalneuralgia, post-herpetic neuralgia, phantom limb pain, fibromyalgia,reflex sympathetic dystrophy, pain, chronic pain, inflammatory pain,post-operative incision pain, post-surgical pain, trauma-related pain,lower back pain, eye pain, tooth pain, complex regional pain syndrome,cancer pain, primary or metastatic bone cancer pain, fracture pain,osteoporotic fracture pain, pain resulting from burn, gout joint pain,pain associated with sickle cell crises, pain associated withtemporomandibular disorders, cirrhosis, hepatitis, neurogenic pain,neuropathic pain, nociceptic pain, visceral pain, menstrual pain,ovarialgia, osteoarthritis pain, rheumatoid arthritis pain, diabeticneuropathy, sciatica, dyspepsia, irritable bowel syndrome, inflammatorybowel disease, Crohn's disease, ileitis, ulcerative colitis, renalcolic, dysmenorrhea, cystitis, interstitial cystitis, menstrual period,labor, menopause, pancreatitis, schizophrenia, depression,post-traumatic stress disorder (“PTSD”), anxiety disorders, autoimmunediabetes, Sjögren's syndrome, multiple sclerosis, overactive bladder,bronchial hyperreactivity, asthma, stroke, bronchitis, bronchodilation,emphysema, chronic obstructive pulmonary disease (“COPD”), inflammatorydermatitis, adenocarcinoma in glandular tissue, blastoma in embryonictissue of organs, carcinoma in epithelial tissue, leukemia in tissuesthat form blood cells, lymphoma in lymphatic tissue, myeloma in bonemarrow, sarcoma in connective or supportive tissue, adrenal cancer,AIDS-related lymphoma, anemia, bladder cancer, bone cancer, braincancer, breast cancer, carcinoid tumors, cervical cancer, chemotherapy,colon cancer, cytopenia, endometrial cancer, esophageal cancer, gastriccancer, head cancer, neck cancer, hepatobiliary cancer, kidney cancer,leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's disease,non-Hodgkin's, nervous system tumors, oral cancer, ovarian cancer,pancreatic cancer, prostate cancer, rectal cancer, skin cancer, stomachcancer, testicular cancer, thyroid cancer, urethral cancer, cancer ofbone marrow, multiple myeloma, tumors that metastasize to the bone,tumors infiltrating the nerve and hollow viscus, tumors near neuralstructures, acne vulgaris, atopic dermatitis, urticaria, keloids,hypertrophic scars and rosacea, endothelial dysfunction, Raynaud'ssyndrome, coronary heart disease (“CHD”), coronary artery disease(“CAD”), heart failure, peripheral arterial disease (“PAD”), diabetes,pulmonary hypertension (“PH”), connective tissue disorder, allergicdermatitis, psoriasis, pruritus, neurogenic cutaneous redness, erythema,sarcoidosis, shock, sepsis, opiate withdrawal syndrome, morphinetolerance, and epilepsy. Additionally, said subject may have a conditionselected from the group consisting of migraine, headache and a painassociated disease or condition, wherein said headache or migraine mayselected from the group consisting of migraine with aura, migrainewithout aura, hemiplegic migraine, cluster headache, migrainousneuralgia, chronic headache, chronic migraine, medication overuseheadache, and tension headache. Also, said subject may have a oculardisorder associated with photophobia that may be selected from the groupconsisting of achromatopsia, aniridia, photophobia caused by ananticholinergic drug, aphakia, buphthalmos, cataracts, cone dystrophy,congenital abnormalities of the eye, viral conjunctivitis, cornealabrasion, corneal dystrophy, corneal ulcer, disruption of the cornealepithelium, ectopia lentis, endophthalmitis, eye trauma caused bydisease, eye trauma caused by injury, eye trauma caused by infection,chalazion, episcleritis, glaucoma, keratoconus, optic nerve hypoplasia,hydrophthalmos, congenital glaucoma iritis, optic neuritis, pigmentdispersion syndrome, pupillary dilation, retinal detachment, scarring ofthe cornea, sclera and uveitis. Further, said subject may have a nervoussystem-related or neurological condition associated with photophobiathat may be selected from the group consisting of autism spectrumdisorders, Chiari malformation, dyslexia, encephalitis, meningitis,subarachnoid hemorrhage, tumor of the posterior cranial fossa,ankylosing spondylitis, albinism, ariboflavinosis, benzodiazepines,chemotherapy, chikungunya, cystinosis, Ehlers-Danlos syndrome, hangover,influenza, infectious mononucleosis, magnesium deficiency, mercurypoisoning, migraine, rabies, and tyrosinemia type II. Additionally, saidsubject may have a photophobia associated disorder that may be selectedfrom the group consisting of migraine with aura, migraine without aura,iritis, uveitis, meningitis, depression, bipolar disorder, clusterheadache or anther trigeminal autonomic cephalalgia (“TAC”) orblepharospasm, depression, agoraphobia and bipolar disorder.

The invention also embraces any of the methods disclosed herein whereinthe method relates to an antibody or antibody fragment that may be ahuman, humanized, or chimerized anti-PACAP antibody or antibodyfragment; and/or wherein the antibody or antibody fragment may a human,humanized, or chimerized anti-PACAP antibody or antibody fragment;and/or wherein the antibody or antibody fragment may be an anti-PACAPantibody or antibody fragment that may comprise the same CDRs as ananti-PACAP antibody that may be selected from Ab1, Ab2, Ab13, Ab14,Ab15, Ab16, Ab17, Ab18, Ab19, Ab1.H, Ab5, Ab7, Ab11, Ab12, Ab4, Ab3,Ab6, Ab8, or Ab9.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention comprise human, humanized, or chimerizedanti-PACAP antibodies or antigen binding fragments thereof, whichcomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 4; a CDR2 sequence consisting of SEQ ID NO: 6;and a CDR3 sequence consisting of SEQ ID NO: 8; and/or (b) a variablelight chain comprising a CDR1 sequence consisting of SEQ ID NO: 24; aCDR2 sequence consisting of SEQ ID NO: 26; and a CDR3 sequenceconsisting of SEQ ID NO: 28. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof may comprise (a) a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 2, and/or (b) avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 22. In amore specific embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof may comprise (a) a variable heavy chain having theamino acid sequence of SEQ ID NO: 2, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 22. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 1, and/or(b) a light chain having the amino acid sequence of SEQ ID NO: 21.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, comprise(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO: 44; a CDR2 sequence consisting of SEQ ID NO: 46; and a CDR3sequence consisting of SEQ ID NO: 48; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO: 64; a CDR2 sequenceconsisting of SEQ ID NO: 66; and a CDR3 sequence consisting of SEQ IDNO: 68. More specifically, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain comprising anamino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99%sequence identity to SEQ ID NO: 42, and/or (b) a variable light chaincomprising an amino acid sequence with at least 80, 85, 90, 95, 96, 97,98, or 99% sequence identity to SEQ ID NO: 62. In another specificembodiment, the anti-PACAP antibodies and antigen binding fragmentsthereof comprise (a) a variable heavy chain having the amino acidsequence of SEQ ID NO: 42, and/or (b) a variable light chain having theamino acid sequence of SEQ ID NO: 62. More specifically, the anti-PACAPantibodies and antigen binding fragments thereof may comprise (a) aheavy chain having the amino acid sequence of SEQ ID NO: 41, and/or (b)a light chain having the amino acid sequence of SEQ ID NO: 61.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 84; a CDR2 sequence consisting of SEQ ID NO:86; and a CDR3 sequence consisting of SEQ ID NO: 88; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:104; a CDR2 sequence consisting of SEQ ID NO: 106; and a CDR3 sequenceconsisting of SEQ ID NO: 108. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise (a) a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 82, and/or (b) avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 102. Inanother specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof may comprise (a) a variable heavy chain havingthe amino acid sequence of SEQ ID NO: 82, and/or (b) a variable lightchain having the amino acid sequence of SEQ ID NO: 102. Morespecifically, the anti-PACAP antibodies and antigen binding fragmentsthereof can comprise (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 81, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 101.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 124; a CDR2 sequence consisting of SEQ ID NO:126; and a CDR3 sequence consisting of SEQ ID NO: 128; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:144; a CDR2 sequence consisting of SEQ ID NO: 146; and a CDR3 sequenceconsisting of SEQ ID NO: 148. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof may comprise (a) a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 122 and/or (b) avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 142. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof may comprise (a) a variable heavy chain having theamino acid sequence of SEQ ID NO: 122, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 142. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 121,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:141.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, preferably human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof, maycomprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 164; a CDR2 sequence consisting of SEQ ID NO:166; and a CDR3 sequence consisting of SEQ ID NO: 168; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:184; a CDR2 sequence consisting of SEQ ID NO: 186; and a CDR3 sequenceconsisting of SEQ ID NO: 188. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise (a) a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 162, and/or (b) avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 182. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 162, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 182. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 161,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:181.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 204; a CDR2 sequence consisting of SEQ ID NO:206; and a CDR3 sequence consisting of SEQ ID NO: 208; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:224; a CDR2 sequence consisting of SEQ ID NO: 226; and a CDR3 sequenceconsisting of SEQ ID NO: 228. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise (a) a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 202 and/or (b) avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 222. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 202, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 222. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 201,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:221.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 244; a CDR2 sequence consisting of SEQ ID NO:246; and a CDR3 sequence consisting of SEQ ID NO: 248; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:264; a CDR2 sequence consisting of SEQ ID NO: 266; and a CDR3 sequenceconsisting of SEQ ID NO: 268. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise (a) a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 242 and/or (b) avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 262. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 242, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 262. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 241,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:261.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 284; a CDR2 sequence consisting of SEQ ID NO:286; and a CDR3 sequence consisting of SEQ ID NO: 288; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:304; a CDR2 sequence consisting of SEQ ID NO: 306; and a CDR3 sequenceconsisting of SEQ ID NO: 308. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 282, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 302. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 282, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 302. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 281,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:301.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 324; a CDR2 sequence consisting of SEQ ID NO:326; and a CDR3 sequence consisting of SEQ ID NO: 328; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:344; a CDR2 sequence consisting of SEQ ID NO: 346; and a CDR3 sequenceconsisting of SEQ ID NO: 348. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 322, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 342. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 322, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 342. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 321,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:341.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 364; a CDR2 sequence consisting of SEQ ID NO:366; and a CDR3 sequence consisting of SEQ ID NO: 368; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:384; a CDR2 sequence consisting of SEQ ID NO: 386; and a CDR3 sequenceconsisting of SEQ ID NO: 388. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 362, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 382. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 362, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 382. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 361,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:381.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 484; a CDR2 sequence consisting of SEQ ID NO:486; and a CDR3 sequence consisting of SEQ ID NO: 488; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:504; a CDR2 sequence consisting of SEQ ID NO: 506; and a CDR3 sequenceconsisting of SEQ ID NO: 508. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 482, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 502. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 482, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 502. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 481,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:501.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 524; a CDR2 sequence consisting of SEQ ID NO:526; and a CDR3 sequence consisting of SEQ ID NO: 528; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:544; a CDR2 sequence consisting of SEQ ID NO: 546; and a CDR3 sequenceconsisting of SEQ ID NO: 548. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 522, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 542. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 522, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 542. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 521,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:541.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 564; a CDR2 sequence consisting of SEQ ID NO:566; and a CDR3 sequence consisting of SEQ ID NO: 568; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:584; a CDR2 sequence consisting of SEQ ID NO: 586; and a CDR3 sequenceconsisting of SEQ ID NO: 588. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 562, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 582. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 562, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 582. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 561,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:581.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 604; a CDR2 sequence consisting of SEQ ID NO:606; and a CDR3 sequence consisting of SEQ ID NO: 608; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:624; a CDR2 sequence consisting of SEQ ID NO: 626; and a CDR3 sequenceconsisting of SEQ ID NO: 628. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 602, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 622. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 602, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 622. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 601,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:621.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 644; a CDR2 sequence consisting of SEQ ID NO:646; and a CDR3 sequence consisting of SEQ ID NO: 648; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:664; a CDR2 sequence consisting of SEQ ID NO: 666; and a CDR3 sequenceconsisting of SEQ ID NO: 668. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 642, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 662. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 642, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 662. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 641,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:661.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 684; a CDR2 sequence consisting of SEQ ID NO:686; and a CDR3 sequence consisting of SEQ ID NO: 688; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:704; a CDR2 sequence consisting of SEQ ID NO: 706; and a CDR3 sequenceconsisting of SEQ ID NO: 708. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 682, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 702. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 682, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 702. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 681,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:701.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 724; a CDR2 sequence consisting of SEQ ID NO:726; and a CDR3 sequence consisting of SEQ ID NO: 728; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:744; a CDR2 sequence consisting of SEQ ID NO: 746; and a CDR3 sequenceconsisting of SEQ ID NO: 748. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 722, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 742. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 722, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 742. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 721,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:741.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 764; a CDR2 sequence consisting of SEQ ID NO:766; and a CDR3 sequence consisting of SEQ ID NO: 768; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:784; a CDR2 sequence consisting of SEQ ID NO: 786; and a CDR3 sequenceconsisting of SEQ ID NO: 788. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof fragment can comprise a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 762, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 782. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 762, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 782. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 761,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:781.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are preferably human, humanized, orchimerized anti-PACAP antibodies and antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 804; a CDR2 sequence consisting of SEQ ID NO:806; and a CDR3 sequence consisting of SEQ ID NO: 808; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:824; a CDR2 sequence consisting of SEQ ID NO: 826; and a CDR3 sequenceconsisting of SEQ ID NO: 828. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 802, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 822. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 802, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 822. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 801,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:821.

The invention also relates to any of the methods disclosed hereinwherein the anti-PACAP antibodies or antibody fragments may be selectedfrom the group consisting of scFvs, camelbodies, nanobodies,Immunoglobulin New Antigen Receptor (“IgNAR”), fragment antigen binding(“Fab”) fragments, Fab′ fragments, MetMab like antibodies, monovalentantibody fragments, and F(ab′)₂ fragments. Additionally, the inventionrelates to any of the methods disclosed herein wherein the anti-PACAPantibody or antibody fragment may substantially or entirely lackN-glycosylation and/or O-glycosylation. Also, the invention pertains toany of the methods disclosed herein wherein the anti-PACAP antibody orantibody fragment may comprise a human constant domain, e.g., that of anIgG1, IgG2, IgG3, or IgG4 antibody.

Another aspect of the invention pertains to any of the methods disclosedherein wherein the anti-PACAP antibody or antibody fragment may comprisean Fc region that may have been modified to alter at least one ofeffector function, half-life, proteolysis, or glycosylation, e.g., theFc region may contain one or more mutations that alters or eliminates N-and/or O-glycosylation.

A further aspect of the invention relates to any of the methodsdisclosed herein wherein the anti-PACAP antibody or antibody fragmentmay bind to PACAP with a binding affinity (K_(D)) of less than or equalto 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹² M, 5×10⁻¹³ M, or 10⁻¹³ M. Also, said anti-PACAP antibody orantibody fragment of any of the methods disclosed herein may bind toPACAP with a binding affinity (K_(D)) of less than or equal to 5×10⁻¹⁰M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, or 10⁻¹² M. Anotherembodiment of the invention pertains any of the methods disclosed hereinwherein the anti-PACAP antibody or antibody fragment may bind to PACAPwith an off-rate (k_(off)) of less than or equal to 5×10⁻⁴ s⁻¹, 10⁻⁴s⁻¹, 5×10⁻⁵ s⁻¹, or 10⁻⁵ s⁻¹.

Moreover, the invention embraces any of the methods disclosed hereinwherein the anti-PACAP antibody or antibody fragment may be directly orindirectly attached to a detectable label or therapeutic agent. Also,the invention relates to any of the methods disclosed herein wherein theanti-PACAP antibody or antibody fragment may bind to PACAP with a KDthat may be less than about 100 nM, less than about 40 nM, less thanabout 1 nM, less than about 100 pM, less than about 50 pM, or less thanabout 25 pM. Also, the invention embraces any of the methods disclosedherein wherein the anti-PACAP antibody or antibody fragment may bind toPACAP with a KD that may be between about 10 pM and about 100 pM. Theinvention further pertains to any of the methods disclosed hereinwherein the method may further comprise administering separately orco-administering another agent, e.g., wherein the other agent may beselected from a chemotherapeutic, an analgesic, an anti-inflammatory, animmunosuppressant, a cytokine, an antiproliferative, an antiemetic or acytotoxin. Also, the invention embraces any of the methods disclosedherein wherein the other therapeutic agent may be an analgesic, and saidanalgesic may be a non-steroidal anti-inflammatory drug (“NSAID”), anopioid analgesic, another antibody or a non-antibody biologic, andfurther wherein said other antibody may be an anti-NGF antibody orantibody fragment; and/or may be an anti-Calcitonin Gene-Related Peptide(“CGRP”) antibody or antibody fragment and/or an anti-CGRP receptorantibody or antibody fragment. The invention also pertains to any of themethods disclosed herein wherein said NSAID may be a cyclooxygenase 1and/or cyclooxygenase 2 inhibitor; and/or wherein said NSAID may beselected from the group consisting of (1) propionic acid derivativesincluding ibuprofen, naproxen, naprosyn, diclofenac, and ketoprofen; (2)acetic acid derivatives including tolmetin and sulindac; (3) fenamicacid derivatives including mefenamic acid and meclofenamic acid; (4)biphenylcarboxylic acid derivatives including diflunisal and flufenisal;and (5) oxicams including piroxim, sudoxicam, and isoxicam. Theinvention further relates to any of the methods disclosed herein whereinsaid opioid analgesic may be selected from the group consisting ofcodeine, dihydrocodeine, diacetylmorphine, hydrocodone, hydromorphone,levorphanol, oxymorphone, alfentanil, buprenorphine, butorphanol,fentanyl, sufentanil, meperidine, methadone, nalbuphine, propoxyphene,pentazocine, and pharmaceutically acceptable salts thereof; and/orwherein the opioid analgesic may be morphine or a morphine derivative orpharmaceutically acceptable salt thereof; and/or wherein the combinedadministration of the opioid analgesic and the anti-PACAP antibody orantigen binding fragment may increase the analgesic effect as comparedto either the opioid analgesic or the anti-PACAP antibody or antigenbinding fragment administered alone.

Furthermore, the invention relates to any of the methods disclosedherein wherein a subject of any of the methods disclosed herein may havepreviously received an anti-CGRP antibody or antibody fragment and/or ananti-CGRP receptor antibody or antibody fragment; and/or wherein saidsubject may be a migraineur who may have not adequately responded toanti-CGRP antibody and/or an anti-CGRP receptor antibody or antibodyfragment treatment; and/or wherein said subject may have previouslyreceived at least one anti-CGRP antibody or antibody fragment and/or ananti-CGRP receptor antibody or antibody fragment administration that mayhave elicited an immune response to the anti-CGRP antibody or antibodyfragment and/or the anti-CGRP receptor antibody or antibody fragment.

Moreover, the invention embraces any of the methods disclosed hereinwherein the antibody or antigen binding fragment thereof of theinvention may ameliorate or treat one or more conditions associated withPACAP expression in a subject in need thereof, said condition may beselected from the group consisting of: migraine with aura, migrainewithout aura, hemiplegic migraines, cluster headaches, migrainousneuralgia, chronic headaches, tension headaches, general headaches, hotflush, photophobia, chronic paroxysmal hemicrania, secondary headachesdue to an underlying structural problem in the head, secondary headachesdue to an underlying structural problem in the neck, cranial neuralgia,sinus headaches, headache associated with sinusitis, allergy-inducedheadaches, allergy-induced migraines, trigeminal neuralgia,post-herpetic neuralgia, phantom limb pain, fibromyalgia, reflexsympathetic dystrophy, pain, chronic pain, inflammatory pain,post-operative incision pain, post-surgical pain, trauma-related pain,lower back pain, eye pain, tooth pain, complex regional pain syndrome,cancer pain, primary or metastatic bone cancer pain, fracture pain,osteoporotic fracture pain, pain resulting from burn, gout joint pain,pain associated with sickle cell crises, pain associated withtemporomandibular disorders, cirrhosis, hepatitis, neurogenic pain,neuropathic pain, nociceptic pain, visceral pain, menstrual pain,ovarialgia, osteoarthritis pain, rheumatoid arthritis pain, diabeticneuropathy, sciatica, dyspepsia, irritable bowel syndrome, inflammatorybowel disease, Crohn's disease, ileitis, ulcerative colitis, renalcolic, dysmenorrhea, cystitis, interstitial cystitis, menstrual period,labor, menopause, pancreatitis, schizophrenia, depression,post-traumatic stress disorder, anxiety disorders, autoimmune diabetes,Sjögren's syndrome, multiple sclerosis, overactive bladder, bronchialhyperreactivity, asthma, stroke, bronchitis, bronchodilation, emphysema,chronic obstructive pulmonary disease (“COPD”), inflammatory dermatitis,adenocarcinoma in glandular tissue, blastoma in embryonic tissue oforgans, carcinoma in epithelial tissue, leukemia in tissues that formblood cells, lymphoma in lymphatic tissue, myeloma in bone marrow,sarcoma in connective or supportive tissue, adrenal cancer, AIDS-relatedlymphoma, anemia, bladder cancer, bone cancer, brain cancer, breastcancer, carcinoid tumors, cervical cancer, chemotherapy, colon cancer,cytopenia, endometrial cancer, esophageal cancer, gastric cancer, headcancer, neck cancer, hepatobiliary cancer, kidney cancer, leukemia,liver cancer, lung cancer, lymphoma, Hodgkin's disease, non-Hodgkin's,nervous system tumors, oral cancer, ovarian cancer, pancreatic cancer,prostate cancer, rectal cancer, skin cancer, stomach cancer, testicularcancer, thyroid cancer, urethral cancer, cancer of bone marrow, multiplemyeloma, tumors that metastasize to the bone, tumors infiltrating thenerve and hollow viscus, tumors near neural structures, acne vulgaris,atopic dermatitis, urticaria, keloids, hypertrophic scars and rosacea,endothelial dysfunction, Raynaud's syndrome, coronary heart disease(“CHD”), coronary artery disease (“CAD”), heart failure, peripheralarterial disease (“PAD”), diabetes, pulmonary hypertension (“PH”),connective tissue disorder, allergic dermatitis, psoriasis, pruritus,neurogenic cutaneous redness, erythema, sarcoidosis, shock, sepsis,opiate withdrawal syndrome, morphine tolerance, and epilepsy.

In another embodiment of the invention, an anti-PACAP antibody orantibody fragment or a method as disclosed herein may inhibit theeffects of PACAP on vasodilation; and/or may inhibit the effects ofPACAP on cAMP production; and/or may inhibit the effects of PACAP on PLCresulting in reduced Ca++ and PLD levels; and/or may inhibit the effectsof PACAP on adenylate cyclase activity; and/or may inhibit the effectsof PACAP on its binding to any or all of PAC1-R, VPAC1-R or VPAC2-R;and/or may inhibit the effects of PACAP on neurodevelopment; may inhibitthe effects of PACAP on neuroprotection; and/or may inhibit the effectsof PACAP on neuromodulation; and/or may inhibit the effects of PACAP onneurogenic inflammation; and/or may inhibit the effects of PACAP onnociception; and/or may modulate the interaction of PACAP with bindingthe cell surface, e.g. via at least one GAG, e.g., wherein at least onesaid GAG may comprise one or more of heparin, chondroitin, keratin, andhyaluronic acid, and further wherein said antibody or antibody fragmentor method may block or inhibit receptor-independent cellular uptake ofPACAP38 and/or PACAP27 and/or may inhibit or may block GAG-dependentuptake of PACAP38 and/or PACAP27 by cells.

A further embodiment of the invention relates to method of therapy orprophylaxis that may comprise the administration of an anti-PACAPantibody or antibody fragment of the invention. Additionally, theinvention pertains to a composition that may be used in human therapythat may contain an anti-PACAP antibody or antibody fragment of theinvention. Said composition may contain another active agent, e.g.,wherein the other agent may selected from a chemotherapeutic, ananalgesic, an anti-inflammatory, an immunosuppressant, a cytokine, anantiproliferative, an antiemetic or a cytotoxin. Wherein said otheragent may be an analgesic, said analgesic may be a NSAID, an opioidanalgesic, another antibody or a non-antibody biologic. When said otheragent may be an analgesic that may be another antibody, the otherantibody may be an anti-NGF antibody or antibody fragment, and/or theother antibody may be an anti-Calcitonin Gene-Related Peptide (“CGRP”)antibody or antibody fragment and/or an anti-CGRP receptor antibody orantibody fragment. Wherein said other agent may be a NSAID, said NSAIDmay be a cyclooxygenase 1 and/or cyclooxygenase 2 inhibitor; and/or saidNSAID may be selected from the group consisting of (1) propionic acidderivatives including ibuprofen, naproxen, naprosyn, diclofenac, andketoprofen; (2) acetic acid derivatives including tolmetin and sulindac;(3) fenamic acid derivatives including mefenamic acid and meclofenamicacid; (4) biphenylcarboxylic acid derivatives including diflunisal andflufenisal; and (5) oxicams including piroxim, sudoxicam, and isoxicam.Wherein said other agent may be an opioid analgesic, said opioidanalgesic may be selected from the group consisting of codeine,dihydrocodeine, diacetylmorphine, hydrocodone, hydromorphone,levorphanol, oxymorphone, alfentanil, buprenorphine, butorphanol,fentanyl, sufentanil, meperidine, methadone, nalbuphine, propoxyphene,pentazocine, and pharmaceutically acceptable salts thereof; and/or saidopioid analgesic may be morphine or a morphine derivative orpharmaceutically acceptable salt thereof; and/or said opioid analgesicand an anti-PACAP antibody or antigen binding fragment according to theinvention may increase the analgesic effect as compared to either theopioid analgesic or the anti-PACAP antibody or antigen binding fragmentadministered alone.

In another embodiment of the invention, an anti-PACAP antibody orfragment or composition according to the invention, wherein theanti-PACAP antibody or fragment and another active agent may be combinedtherewith or may be administered in combination, may elicit asynergistic or additive effect on the treatment or prevention of a PACAPassociated effect, e.g., migraine or on pain. Another embodiment of theinvention additionally embraces an anti-PACAP antibody or fragment orcomposition according the invention that may be used in therapy ordiagnosis, e.g., migraine treatment or prophylaxis. A further embodimentof the invention relates to an anti-PACAP antibody or fragment orcomposition according to the invention that may be used for treating oneor more of hot flush, migraine with or without aura, hemiplegicmigraine, cluster headache, migrainous neuralgia, chronic headache,chronic migraine, medication overuse headache or tension headache.

Another embodiment of the invention pertains an anti-PACAP antibody orfragment or composition according to the invention that may be used forameliorating, controlling, reducing incidence of, or delaying thedevelopment or progression of headache, e.g., migraine with or withoutaura, hemiplegic migraine, cluster headache, migrainous neuralgia,chronic headache, chronic migraine, medication overuse headache ortension headache. In an embodiment of the invention, the use may be fora subject who may have previously received or may be receiving ananti-CGRP antibody or antibody fragment and/or an anti-CGRP receptorantibody or antibody fragment, and further wherein said subject may be amigraineur who may not have adequately responded to anti-CGRP antibodyor antibody fragment and/or anti-CGRP receptor antibody or antibodyfragment treatment; and/or wherein said subject may have previouslyreceived at least one anti-CGRP antibody or antibody fragment and/oranti-CGRP receptor antibody or antibody fragment administration that mayhave elicited an immune response to the anti-CGRP antibody or antibodyfragment and/or anti-CGRP receptor antibody or antibody fragment.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1A-1B provides the polypeptide sequences of the heavy chainvariable region for antibodies Ab1, Ab1.H, Ab2, Ab13, Ab14, Ab15, Ab16,Ab17, Ab18, Ab19, Ab5, Ab7, Ab11, Ab12, Ab4, Ab3, Ab6, Ab8, and Ab9 (SEQID NOs: 2; 42; 82; 122; 162; 202; 242; 282; 322; 362; 482; 522; 562;602; 642; 682; 722; 762; and 802, respectively) aligned by their FRs andCDRs.

FIG. 2A-2B provides the polypeptide sequences of the light chainvariable region for antibodies Ab1, Ab1.H, Ab2, Ab13, Ab14, Ab15, Ab16,Ab17, Ab18, Ab19, Ab5, Ab7, Ab11, Ab12, Ab4, Ab3, Ab6, Ab8, and Ab9 (SEQID NOs: 22; 62; 102; 142; 182; 222; 262; 302; 342; 382; 502; 542; 582;622; 662; 702; 742; 782; and 822, respectively) aligned by their FRs,and CDRs.

FIG. 3A-3F provides the polynucleotide sequences encoding the heavychain variable region for antibodies Ab1, Ab1.H, Ab2, Ab13, Ab14, Ab15,Ab16, Ab17, Ab18, Ab19, Ab5, Ab7, Ab11, Ab12, Ab4, Ab3, Ab6, Ab8, andAb9 (SEQ ID NOs: 12; 52; 92; 132; 172; 212; 252; 292; 332; 372; 492;532; 572; 612; 652; 692; 732; 772; and 812, respectively) aligned bytheir FRs, and CDRs.

FIG. 4A-4E provides the polynucleotide sequences encoding the lightchain variable region for antibodies Ab1, Ab1.H, Ab2, Ab13, Ab14, Ab15,Ab16, Ab17, Ab18, Ab19, Ab5, Ab7, Ab11, Ab12, Ab4, Ab3, Ab6, Ab8, andAb9 (SEQ ID NOs: 32; 72; 112; 152; 192; 232; 272; 312; 352; 392; 512;552; 592; 632; 672; 712; 752; 792; and 832, respectively) aligned bytheir FRs, and CDRs.

FIG. 5 provides the polypeptide sequence coordinates for certainantibody heavy chain protein sequence features including the variableregion and complementarity determining regions (CDRs) of the heavy chainfor antibodies Ab1, Ab1.H, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11,Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, and Ab19.

FIG. 6 provides the polypeptide sequence coordinates for certainantibody heavy chain protein sequence features including the constantregion and framework regions (FR) of the heavy chain for antibodies Ab1,Ab1.H, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13, Ab14,Ab15, Ab16, Ab17, Ab18, and Ab19.

FIG. 7 provides the polypeptide sequence coordinates for certainantibody light chain protein sequence features including the variableregion and complementarity determining regions (CDRs) of the light chainfor antibodies Ab1, Ab1.H, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11,Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, and Ab19.

FIG. 8 provides the polypeptide sequence coordinates for certainantibody light chain protein sequence features including the constantregion and framework regions (FR) of the light chain for antibodies Ab1,Ab1.H, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13, Ab14,Ab15, Ab16, Ab17, Ab18, and Ab19.

FIG. 9 provides the polynucleotide sequence coordinates for certainantibody heavy chain DNA sequence features including the variable regionand complementarity determining regions (CDRs) of the heavy chain forantibodies Ab1, Ab1.H, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11,Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, and Ab19.

FIG. 10 provides the polynucleotide sequence coordinates for certainantibody heavy chain DNA sequence features including the constant regionand framework regions (FR) of the heavy chain for antibodies Ab1, Ab1.H,Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13, Ab14, Ab15,Ab16, Ab17, Ab18, and Ab19.

FIG. 11 provides the polynucleotide sequence coordinates for certainantibody light chain DNA sequence features including the variable regionand complementarity determining regions (CDRs) of the light chain forantibodies Ab1, Ab1.H, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11,Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, and Ab19.

FIG. 12 provides the polynucleotide sequence coordinates for certainantibody light chain DNA sequence features including the constant regionand framework regions (FR) of the light chain for antibodies Ab1, Ab1.H,Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13, Ab14, Ab15,Ab16, Ab17, Ab18, and Ab19.

FIG. 13 provides representative data showing a reduction in vasodilationobtained by administering Ab1.H following PACAP38 administration in arabbit model, relative to a vehicle control, obtained following theprotocol in Example 7 infra.

FIG. 14 provides representative data showing a reduction in vasodilationobtained by administering Ab10 following PACAP38 administration in arabbit model, relative to an isotype antibody control, obtainedfollowing the protocol in Example 8 infra.

FIG. 15A provides epitope binning data for labeled Ab1 and unlabeledAb10 obtained following the protocol in Example 9 infra.

FIG. 15B provides epitope binning data for unlabeled Ab1 and labeledAb10 obtained following the protocol in Example 9 infra.

FIG. 16 provides representative data showing the in vivo effect of theadministration of PACAP and an anti-PACAP antibody Ab1.H in a rodentphotophobia model, which model detects the amount of time treatedanimals (mice) spend in the light per 5 minute intervals compared toappropriate control animals obtained following the protocol in Example11 infra.

FIG. 17 provides representative data showing the in vivo effect of theadministration of PACAP and anti-PACAP antibody Ab1.H in a rodentphotophobia animal model, which detects the average amount of timetreated animals (mice) spend in the light compared to appropriatecontrol animals obtained following the protocol in Example 11 infra.

FIG. 18 provides representative data showing the in vivo effect of theadministration of PACAP and an anti-PACAP antibody Ab10.H in a rodentphotophobia model, which model detects the amount of time treatedanimals (mice) spend in the light compared to appropriate controlanimals obtained following the protocol in Example 11 infra.

FIG. 19A-J provides representative data showing Ab1.H (FIG. 19A), Ab3.H(FIG. 19B), Ab4.H (FIG. 19C), Ab5.H (FIG. 19D), Ab9.H (FIG. 19E), Ab12.H(FIG. 19F), Ab10 (FIG. 19G), Ab10.H (FIG. 19H), Ab22 (FIG. 19I), andAb23 (FIG. 19J) binding to PAC1-R-expressing PC-12 cells in the presenceof PACAP38 obtained following the protocol in Example 6 infra.

DETAILED DESCRIPTION Definitions

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by the appendedclaims. As used herein the singular forms “a”, “and”, and “the” includeplural referents unless the context clearly dictates otherwise. Thus,for example, reference to “a cell” includes a plurality of such cellsand reference to “the protein” includes reference to one or moreproteins and equivalents thereof known to those skilled in the art, andso forth. All technical and scientific terms used herein have the samemeaning as commonly understood to one of ordinary skill in the art towhich this invention belongs unless clearly indicated otherwise.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP):

As used herein, unless stated otherwise PACAP includes any mammalianform of PACAP, and in particular encompasses the following Homo sapiensPACAP27 and Homo sapiens PACAP38 amino acid sequences:

PACAP38:

HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 1241), wherein theC-terminal lysine is amidated; but also any mutants, splice variants,isoforms, orthologs, homologs, and variants of this sequence.

PACAP27:

HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 1242), wherein the C-terminalleucine is amidated; but also any mutants, splice variants, isoforms,orthologs, homologs, and variants of this sequence.

“Photophobia” herein refers to a symptom of abnormal intolerance tovisual perception of light, sometimes additionally defined by abnormalor irrational fear of light, or by presence of actual physicalphotosensitivity of the eyes. In the present invention photophobiaincludes in particular light aversion associated with migraine, clusterheadaches and other neurological causes of light aversive behavior thatcan trigger a migraine or cluster headache. Patients/subjects candevelop photophobia as a result of several different medical conditions,related to the eye or the nervous system. Photophobia can be caused byan increased response to light starting at any step in the visual systemsuch as: (i) too much light entering the eye, (ii) too much light canenter the eye if it is damaged, such as with corneal abrasion andretinal damage, or if a pupil(s) is unable to normally constrict (seenwith damage to the oculomotor nerve), (iii) overstimulation of thephotoreceptors in the retina, (iv) excessive electric impulses to theoptic nerve, and (v) excessive response in the central nervous system.

“Effective treatment or prevention of photophobia” herein refers toinhibiting light aversive behavior or photophobia or inhibiting theonset of light aversive behavior or photophobia in a subject in needthereof, e.g., a subject having an active migraine attack or clusterheadache or a subject prone to migraine or cluster headaches, or one ofthe other photophobia-associated disorders identified herein afteradministration of an effective amount of an anti-PACAP antibody orantigen binding fragment thereof according to the invention. Thetreatment may be effected as a monotherapy or in association withanother active agent such as topiramate or dihydroergotamine by way ofexample.

The term “migraine” refers to a complex and disabling neurologicaldisorder that may progress during four stages: prodrome, aura, headache,and postdrome. A migraine is defined by the International HeadacheSociety as a headache that lasts for 4-72 hours and is characterized byat least two of the following: unilateral localization, pulsatingquality, moderate to severe pain intensity; and aggravation by movementsuch as walking. In addition, the headache must be accompanied by atleast one of the following: nausea and/or vomiting, photophobia, orphonophobia. A migraine may also be accompanied by aura, which typicallyprecedes the deadline during the premonition or prodrome phase, andoften results in visual changes, e.g., a scintillating scotoma thatmoves across the visual field. The prodrome may also be accompanied byother symptoms, e.g., fatigue, gastrointestinal issues, and moodchanges. A migraineur is often incapacitated for extended periods oftime. The postdrome is the final phase and occurs after the attack,during which time the migraineur may feel exhausted or mildly euphoric.

The term “headache” refers to pain in any region of the head. Headachesmay occur on one or both sides of the head, be isolated to a certainlocation, radiate across the head from one point, or have a vise-likequality. A headache may be a sharp pain, throbbing sensation or dullache. Headaches may appear gradually or suddenly, and they may last lessthan an hour or for several days.

The term “pain associated disease or condition” refers to any disease orcondition defined, in whole or in part, by acute and/or chronic pain.Pain is generally defined as an unpleasant sensory and emotionalexperience associated with actual or potential tissue damage, ordescribed in terms of such damage. Pain may be classified as neurogenic,neuropathic, inflammatory, or nociceptic.

The term “opioid analgesic” herein refers to all drugs, natural orsynthetic, with morphine-like actions. The synthetic and semi-syntheticopioid analgesics are derivatives of five chemical classes of compound:phenanthrenes; phenylheptylamines; phenylpiperidines; morphinans; andbenzomorphans, all of which are within the scope of the term. Exemplaryopioid analgesics include codeine, dihydrocodeine, diacetylmorphine,hydrocodone, hydromorphone, levorphanol, oxymorphone, alfentanil,buprenorphine, butorphanol, fentanyl, sufentanil, meperidine, methadone,nalbuphine, propoxyphene, and pentazocine, or pharmaceuticallyacceptable salts thereof.

The term “NSAID” refers to a non-steroidal anti-inflammatory compound.NSAIDs are categorized by virtue of their ability to inhibitcyclooxygenase. Cyclooxygenase 1 and cyclooxygenase 2 are two majorisoforms of cyclooxygenase and most standard NSAIDs are mixed inhibitorsof the two isoforms. Most standard NSAIDs fall within one of thefollowing five structural categories: (1) propionic acid derivatives,such as ibuprofen, naproxen, naprosyn, diclofenac, and ketoprofen; (2)acetic acid derivatives, such as tolmetin and sulindac; (3) fenamic acidderivatives, such as mefenamic acid and meclofenamic acid; (4)biphenylcarboxylic acid derivatives, such as diflunisal and flufenisal;and (5) oxicams, such as piroxim, sudoxicam, and isoxicam. Another classof NSAID has been described that selectively inhibit cyclooxygenase 2.COX-2 inhibitors have been described, e.g., in U.S. Pat. Nos. 5,616,601;5,604,260; 5,593,994; 5,550,142; 5,536,752; 5,521,213; 5,475,995;5,639,780; 5,604,253; 5,552,422; 5,510,368; 5,436,265; 5,409,944; and5,130,311, all of which are hereby incorporated by reference. Certainexemplary COX-2 inhibitors include celecoxib (SC-58635), DUP-697,flosulide (CGP-28238), meloxicam, 6-methoxy-2 naphthylacetic acid(6-MNA), rofecoxib, MK-966, nabumetone (prodrug for 6-MNA), nimesulide,NS-398, SC-5766, SC-58215, T-614; or combinations thereof.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: improvement in any aspect of PACAP-related conditionssuch as migraine or headache. For example in the context of headache ormigraine treatment this includes lessening severity, alleviation of painintensity, and other associated symptoms, reducing frequency ofrecurrence, increasing the quality of life of those suffering from theheadache, and decreasing dose of other medications required to treat theheadache. For migraine, other associated symptoms include, but are notlimited to, nausea, vomiting, and sensitivity to light, sound, and/ormovement. For cluster headache, other associated symptoms include, butare not limited to swelling under or around the eyes, excessive tears,red eye, rhinorrhea or nasal congestion, and red flushed face.

“Reducing incidence” or “prophylaxis” or “prevention” means any ofreducing severity for a particular disease, condition, symptom, ordisorder (the terms disease, condition, and disorder are usedinterchangeably throughout the application). Reduction in severityincludes reducing drugs and/or therapies generally used for thecondition by, for example, reducing the need for, amount of, and/orexposure to drugs or therapies. Reduction in severity also includesreducing the duration, and/or frequency of the particular condition,symptom, or disorder (including, for example, delaying or increasingtime to next episodic attack in an individual).

“Ameliorating” headache or one or more symptoms of headache or migraineor other PACAP-related condition means a lessening or improvement of oneor more symptoms of the condition, e.g., headache or migraine ascompared to not administering an anti-PACAP antagonist antibody.“Ameliorating” also includes shortening or reduction in duration of asymptom.

As used herein, “controlling headache” or “controlling migraine” or“controlling” another PACAP-related condition refers to maintaining orreducing severity or duration of one or more symptoms of the condition,e.g., headache or migraine or frequency of headache or migraine attacksin an individual (as compared to the level before treatment). Forexample, the duration or severity of head pain, or frequency of attacksis reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 100% in the individual as compared to the level beforetreatment. The reduction in the duration or severity of head pain, orfrequency of attacks can last for any length of time, e.g., 2 weeks, 4weeks (1 month), 8 weeks (2 months), 16 weeks (3 months), 4 months, 5months, 6 months, 9 months, 12 months, etc.

As used therein, “delaying” the development of a PACAP-related conditionsuch as migraine or headache means to defer, hinder, slow, retard,stabilize, and/or postpone progression of the condition or disease. Thisdelay can be of varying lengths of time, depending on the history of thecondition or disease and/or individuals being treated. As is evident toone skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developheadache (e.g., migraine). A method that “delays” development of thesymptom is a method that reduces probability of developing the symptomin a given time frame and/or reduces extent of the symptoms in a giventime frame, when compared to not using the method. Such comparisons aretypically based on clinical studies, using a statistically significantnumber of subjects.

“Development” or “progression” of a PACAP-related condition such asmigraine or headache, preferably a condition associated with photophobiaor light aversion, means initial manifestations and/or ensuingprogression of the disorder or symptom or side effect of such disordersuch as photophobia or light aversion. Development of headache ormigraine can be detectable and assessed using standard clinicaltechniques as well known in the art. However, development also refers toprogression that may be undetectable. For purpose of this invention,development, or progression refers to the biological course of thesymptoms. “Development” includes occurrence, recurrence, and onset. Asused herein “onset” or “occurrence” of a condition such as headache ormigraine includes initial onset and/or recurrence.

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toeffect beneficial or desired results, e.g., an amount sufficient totreat or ameliorate or prevent PACAP associate photophobia or lightaversion. For prophylactic use, beneficial or desired results includeresults such as eliminating or reducing the risk, lessening theseverity, or delaying the outset of the disease, including biochemical,histological, and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. For therapeutic use, beneficial or desiredresults include clinical results such as reducing pain intensity,duration, or frequency of headache attack, and decreasing one or moresymptoms resulting from headache (biochemical, histological, and/orbehavioral), including its complications and intermediate pathologicalphenotypes presenting during development of the disease, increasing thequality of life of those suffering from the disease, decreasing the doseof other medications required to treat the disease, enhancing effect ofanother medication, and/or delaying the progression of the disease ofpatients. For instance, clinical results include reducing the sensationof photophobia, light aversion, or avoidance and sensitivity to light.An effective dosage can be administered in one or more administrations.For purposes of this invention, an effective dosage of drug, compound,or pharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective dosage of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective dosage” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

A “suitable host cell” or “host cell” generally includes any cellwherein the subject anti-PACAP antibodies and antigen binding fragmentsthereof can be produced recombinantly using techniques and materialsreadily available. For example, the anti-PACAP antibodies and antigenbinding fragments thereof of the present invention can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells (e.g., yeast), and cultured higher eukaryoticcells (including cultured cells of multicellular organisms),particularly cultured mammalian cells, e.g., human or non-humanmammalian cells. In an exemplary embodiment these antibodies may beexpressed in CHO cells. Techniques for manipulating cloned DNA moleculesand introducing exogenous DNA into a variety of host cells are disclosedby Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989),and Current Protocols in Molecular Biology, Ausubel et al., editors, NewYork, N.Y.: Green and Wiley and Sons (1993).

In some exemplary embodiments the antibodies may be expressed in matingcompetent yeast, e.g., any haploid, diploid or tetraploid yeast that canbe grown in culture. Yeast useful in fermentation expression methods mayexist in a haploid, diploid, or other polyploid form. The cells of agiven ploidy may, under appropriate conditions, proliferate for anindefinite number of generations in that form. Diploid cells can alsosporulate to form haploid cells. Sequential mating can result intetraploid strains through further mating or fusion of diploid strains.The present invention contemplates the use of haploid yeast, as well asdiploid or other polyploid yeast cells produced, for example, by matingor spheroplast fusion. By way of example, such yeast may include membersof the Saccharomycetaceae family, which includes the genera Arxiozyma;Ascobotryozyma; Citeromyces; Debaryomyces; Dekkera; Eremothecium;Issatchenkia; Kazachstania; Kluyveromyces; Kodamaea; Lodderomyces;Pachysolen; Pichia; Saccharomyces; Saturnispora; Tetrapisispora;Torulaspora; Williopsis; and Zygosaccharomyces. Other types of yeastpotentially useful in the invention include Yarrowia; Rhodosporidium;Candida; Hansenula; Filobasium; Sporidiobolus; Bullera; Leucosporidiumand Filobasidella.

In a preferred exemplary embodiment of the invention, the matingcompetent yeast used for antibody expression may comprise a member ofthe genus Pichia. In a further preferred exemplary embodiment of theinvention, the mating competent yeast of the genus Pichia is one of thefollowing species: Pichia pastoris, Pichia methanolica, and Hansenulapolymorpha (Pichia angusta). In a particularly preferred embodiment ofthe invention, the mating competent yeast of the genus Pichia is thespecies Pichia pastoris.

A “selectable marker” herein refers to a gene or gene fragment thatconfers a growth phenotype (physical growth characteristic) on a cellreceiving that gene as, for example through a transformation event. Theselectable marker allows that cell to survive and grow in a selectivegrowth medium under conditions in which cells that do not receive thatselectable marker gene cannot grow. Selectable marker genes generallyfall into several types, including positive selectable marker genes suchas a gene that confers on a cell resistance to an antibiotic or otherdrug, temperature when two temperature sensitive (“ts”) mutants arecrossed or a is mutant is transformed; negative selectable marker genessuch as a biosynthetic gene that confers on a cell the ability to growin a medium without a specific nutrient needed by all cells that do nothave that biosynthetic gene, or a mutagenized biosynthetic gene thatconfers on a cell inability to grow by cells that do not have the wildtype gene; and the like. Suitable markers include but are not limitedto: ZEO; G418; LYS3; MET1; MET3a; ADE1; ADE3; URA3; and the like.

An “expression vector” herein refers to DNA vectors containing elementsthat facilitate manipulation for the expression of a foreign proteinwithin the target host cell, e.g., a bacterial, insect, yeast, plant,amphibian, reptile, avian, or mammalian cell, and most typically a yeastor mammalian cell, e.g., a CHO cell. Conveniently, manipulation ofsequences and production of DNA for transformation is first performed ina bacterial host, e.g. E. coli, and usually vectors will includesequences to facilitate such manipulations, including a bacterial originof replication and appropriate bacterial selection marker. Selectionmarkers encode proteins necessary for the survival or growth oftransformed host cells grown in a selective culture medium. Host cellsnot transformed with the vector containing the selection gene will notsurvive in the culture medium. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, (b)complement auxotrophic deficiencies, or (c) supply critical nutrientsnot available from complex media. Exemplary vectors and methods fortransformation of yeast are described, for example, in Burke, D.,Dawson, D., & Stearns, T., Methods in yeast genetics: a Cold SpringHarbor Laboratory course manual, Plainview, N.Y.: Cold Spring HarborLaboratory Press (2000). Expression vectors for use in the methods ofthe invention may include yeast or mammalian specific sequences,including a selectable auxotrophic or drug marker for identifyingtransformed host strains. A drug marker may further be used to amplifycopy number of the vector in a yeast host cell.

The polypeptide coding sequence of interest is operably linked totranscriptional and translational regulatory sequences that provide forexpression of the polypeptide in the desired host cells, e.g., yeast ormammalian cells. These vector components may include, but are notlimited to, one or more of the following: an enhancer element, apromoter, and a transcription termination sequence. Sequences for thesecretion of the polypeptide may also be included, e.g. a signalsequence, and the like. An origin of replication, e.g., a yeast originof replication, is optional, as expression vectors are often integratedinto the host cell genome. In one embodiment of the invention, thepolypeptide of interest is operably linked, or fused, to sequencesproviding for optimized secretion of the polypeptide from yeast diploidcells.

Nucleic acids are “operably linked” when placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for asignal sequence is operably linked to DNA for a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading frame. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites oralternatively via a PCR/recombination method familiar to those skilledin the art (GATEWAY® Technology; Invitrogen, Carlsbad Calif.). If suchsites do not exist, the synthetic oligonucleotide adapters or linkersare used in accordance with conventional practice.

Promoters are untranslated sequences located upstream (5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of particular nucleic acidsequences to which they are operably linked. Such promoters fall intoseveral classes: inducible, constitutive, and repressible promoters(that increase levels of transcription in response to absence of arepressor). Inducible promoters may initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, e.g., the presence or absence of a nutrient or achange in temperature.

The promoter fragment may also serve as the site for homologousrecombination and integration of the expression vector into the samesite in the host cell, e.g., yeast cell, genome; alternatively, aselectable marker may be used as the site for homologous recombination.Pichia transformation is described in Cregg et al., Mol. Cell. Biol.,5:3376-3385 (1985). Suitable promoters for use in different eukaryoticand prokaryotic cells are well known and commercially available.

The polypeptides of interest may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, e.g. a signal sequence or other polypeptide having aspecific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the polypeptide coding sequence that isinserted into the vector. The heterologous signal sequence selectedpreferably is one that is recognized and processed through one of thestandard pathways available within the host cell, e.g., a mammaliancell, an insect cell, or a yeast cell. Additionally, these signalpeptide sequences may be engineered to provide for enhanced secretion inexpression systems. Secretion signals of interest also include mammalianand yeast signal sequences, which may be heterologous to the proteinbeing secreted, or may be a native sequence for the protein beingsecreted. Signal sequences include pre-peptide sequences, and in someinstances may include propeptide sequences. Many such signal sequencesare known in the art, including the signal sequences found onimmunoglobulin chains, e.g., K28 preprotoxin sequence, PHA-E, FACE,human MCP-1, human serum albumin signal sequences, human Ig heavy chain,human Ig light chain, and the like. For example, see Hashimoto et. al.,Protein Eng., 11(2):75 (1998); and Kobayashi et. al., TherapeuticApheresis, 2(4):257 (1998)).

Transcription may be increased by inserting a transcriptional activatorsequence into the vector. These activators are cis-acting elements ofDNA, usually about from 10 to 300 bp, which act on a promoter toincrease its transcription. Transcriptional enhancers are relativelyorientation and position independent, having been found 5′ and 3′ to thetranscription unit, within an intron, as well as within the codingsequence itself. The enhancer may be spliced into the expression vectorat a position 5′ or 3′ to the coding sequence, but is preferably locatedat a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells may also containsequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from 3′ tothe translation termination codon, in untranslated regions of eukaryoticor viral DNAs or cDNAs. These regions contain nucleotide segmentstranscribed as polyadenylated fragments in the untranslated portion ofthe mRNA.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques orPCR/recombination methods. Isolated plasmids or DNA fragments arecleaved, tailored, and re-ligated in the form desired to generate theplasmids required or via recombination methods. For analysis to confirmcorrect sequences in plasmids constructed, the ligation mixtures areused to transform host cells, and successful transformants selected byantibiotic resistance (e.g. ampicillin or Zeocin) where appropriate.Plasmids from the transformants are prepared, analyzed by restrictionendonuclease digestion, and/or sequenced.

As an alternative to restriction and ligation of fragments,recombination methods based on specific attachment (“att”) sites andrecombination enzymes may be used to insert DNA sequences into a vector.Such methods are described, for example, by Landy, Ann. Rev. Biochem.,58:913-949 (1989); and are known to those of skill in the art. Suchmethods utilize intermolecular DNA recombination that is mediated by amixture of lambda and E. coli-encoded recombination proteins.Recombination occurs between att sites on the interacting DNA molecules.For a description of att sites see Weisberg and Landy, Site-SpecificRecombination in Phage Lambda, in Lambda II, p. 211-250, Cold SpringHarbor, N.Y.: Cold Spring Harbor Press (1983). The DNA segments flankingthe recombination sites are switched, such that after recombination, theatt sites are hybrid sequences comprised of sequences donated by eachparental vector. The recombination can occur between DNAs of anytopology.

Att sites may be introduced into a sequence of interest by ligating thesequence of interest into an appropriate vector; generating a PCRproduct containing att B sites through the use of specific primers;generating a cDNA library cloned into an appropriate vector containingatt sites; and the like.

Folding, as used herein, refers to the three-dimensional structure ofpolypeptides and proteins, where interactions between amino acidresidues act to stabilize the structure. While non-covalent interactionsare important in determining structure, usually the proteins of interestwill have intra- and/or intermolecular covalent disulfide bonds formedby two cysteine residues. For naturally occurring proteins andpolypeptides or derivatives and variants thereof, the proper folding istypically the arrangement that results in optimal biological activity,and can conveniently be monitored by assays for activity, e.g. ligandbinding, enzymatic activity, etc.

In some instances, for example where the desired product is of syntheticorigin, assays based on biological activity will be less meaningful. Theproper folding of such molecules may be determined on the basis ofphysical properties, energetic considerations, modeling studies, and thelike.

The expression host may be further modified by the introduction ofsequences encoding one or more enzymes that enhance folding anddisulfide bond formation, i.e. foldases, chaperonins, etc. Suchsequences may be constitutively or inducibly expressed in the yeast hostcell, using vectors, markers, etc. as known in the art. Preferably thesequences, including transcriptional regulatory elements sufficient forthe desired pattern of expression, are stably integrated in the yeastgenome through a targeted methodology.

For example, the eukaryotic protein disulfide isomerase (“PDI”) is notonly an efficient catalyst of protein cysteine oxidation and disulfidebond isomerization, but also exhibits chaperone activity. Co-expressionof PDI can facilitate the production of active proteins having multipledisulfide bonds. Also of interest is the expression of immunoglobulinheavy chain binding protein (“BIP”); cyclophilin; and the like. In oneembodiment of the invention, each of the haploid parental strainsexpresses a distinct folding enzyme, e.g. one strain may express BIP,and the other strain may express PDI or combinations thereof.

Cultured mammalian cells are also preferred exemplary hosts forproduction of the disclosed anti-PACAP antibodies and antigen bindingfragments thereof. As mentioned CHO cells are particularly suitable forexpression of antibodies. Many procedures are known in the art formanufacturing monoclonal antibodies in mammalian cells. (See, Galfre, G.and Milstein, C., Methods Enzym., 73:3-46, 1981; Basalp et al., Turk. J.Biol., 24:189-196, 2000; Wurm, F. M., Nat. Biotechnol., 22:1393-1398,2004; and Li et al., mAbs, 2(5):466-477, 2010). As mentioned in furtherdetail infra, common host cell lines employed in mammalian monoclonalantibody manufacturing schemes include, but are not limited to, humanembryonic retinoblast cell line PER.C6® (Crucell N. V., Leiden, TheNetherlands), NS0 murine myeloma cells (Medical Research Council,London, UK), CV1 monkey kidney cell line, 293 human embryonic kidneycell line, BHK baby hamster kidney cell line, VERO African green monkeykidney cell line, human cervical carcinoma cell line HELA, MDCK caninekidney cells, BRL buffalo rat liver cells, W138 human lung cells, HepG2human liver cells, MMT mouse mammary tumor cells, TRI cells, MRC5 cells,Fs4 cells, myeloma or lymphoma cells, or Chinese Hamster (Cricetulusgriseus) Ovary (CHO) cells, and the like. Many different subclones orsub-cell lines of CHO cells known in the art that are useful andoptimized for production of recombinant monoclonal antibodies, such asthe DP12 (CHO K1 dhfr−) cell line, NS0 cells are a non-Ig secreting,non-light chain-synthesizing subclone of NS-1 cells that are resistantto azaguanine. Other Chinese Hamster and CHO cells are commerciallyavailable (from ATCC, etc.), including CHO-DXB11 (CHO-DUKX), CHO-pro3,CHO-DG44, CHO 1-15, CHO DP-12, Lec2, M1WT3, Lec8, pgsA-745, and thelike, all of which are genetically altered to optimize the cell line forvarious parameters. Monoclonal antibodies are commonly manufacturedusing a batch fed method whereby the monoclonal antibody chains areexpressed in a mammalian cell line and secreted into the tissue culturemedium in a bioreactor. Medium (or feed) is continuously supplied to thebioreactor to maximize recombinant protein expression. Recombinantmonoclonal antibody is then purified from the collected media. In somecircumstances, additional steps are needed to reassemble the antibodiesthrough reduction of disulfide bonds, etc. Such production methods canbe scaled to be as large as 10,000 L in a single batch or more. It isnow routine to obtain as much as 20 pg/cell/day through the use of suchcell lines and methodologies, providing titers as high as 10 g/L ormore, amounting to 15 to 100 kg from bioreactors of 10 kL to 25 kL. (Liet al., 2010). Various details of this production methodology, includingcloning of the polynucleotides encoding the antibodies into expressionvectors, transfecting cells with these expression vectors, selecting fortransfected cells, and expressing and purifying the recombinantmonoclonal antibodies from these cells are provided below.

For recombinant production of an anti-PACAP antibody or antigen bindingfragment in mammalian cells, nucleic acids encoding the antibody orfragment thereof are generally inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated or synthesized usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to DNAs encoding the heavy and lightchains of the antibody). The vector components generally include, butare not limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Selection ofpromoters, terminators, selectable markers, vectors, and other elementsis a matter of routine design within the level of ordinary skill in theart. Many such elements are known in the art and are available throughcommercial suppliers.

The antibodies of this invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The homologous or heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. In mammalian cell expression,mammalian signal sequences as well as viral secretory leaders, forexample, the herpes simplex gD signal, are available.

Such expression vectors and cloning vectors will generally contain anucleic acid sequence that enables the vector to replicate in one ormore selected host cells. Typically, in cloning vectors this sequence isone that enables the vector to replicate independently of the hostchromosomal DNA, and includes origins of replication or autonomouslyreplicating sequences. Such sequences are well known for a variety ofbacteria, yeast, and viruses, e.g., the origin of replication from theplasmid pBR322 is suitable for most Gram-negative bacteria, the 2 muplasmid origin is suitable for yeast, and various viral origins (SimianVirus 40 (“SV40”), polyoma, adenovirus, vesicular stomatitis virus(“VSV”), or bovine papillomavirus (“BPV”) are useful for cloning vectorsin mammalian cells. Generally, the origin of replication component isnot needed for mammalian expression vectors (the SV40 origin maytypically be used only because it contains the early promoter).

These vectors will also typically contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Drug selection is generally used to select for culturedmammalian cells into which foreign DNA has been inserted. Such cells arecommonly referred to as “transfectants”. Cells that have been culturedin the presence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.”Examples of such dominant selection use the drugs neomycin, mycophenolicacid, and hygromycin. An exemplary selectable marker is a gene encodingresistance to the antibiotic neomycin. Selection is carried out in thepresence of a neomycin-type drug, such as G-418 or the like. Those cellsthat are successfully transformed with a heterologous gene produce aprotein conferring drug resistance and thus survive the selectionregimen.

Selection systems can also be used to increase the expression level ofthe gene of interest, a process referred to as “amplification.”Amplification of transfectants typically occurs by culturing the cellsin the presence of a low level of the selective agent and thenincreasing the amount of selective agent to select for cells thatproduce high levels of the products of the introduced genes. Exemplarysuitable selectable markers for mammalian cells are those that enablethe identification of cells competent to take up the antibody nucleicacid, such as dihydrofolate reductase (“DHFR”), thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, an amplifiable selectable marker for mammalian cells isdihydrofolate reductase, which confers resistance to methotrexate. Otherdrug resistance genes (e.g. hygromycin resistance, multi-drugresistance, puromycin acetyltransferase) can also be used. Cellstransformed with the DHFR selection gene are first identified byculturing all of the transformants in a culture medium that containsmethotrexate (“MTX”), a competitive antagonist of DHFR. An appropriatehost cell when wild-type DHFR is employed is the Chinese hamster ovary(“CHO”) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding antibody, wild-type DHFR protein, and another selectable markersuch as aminoglycoside 3′-phosphotransferase (“APH”) can be selected bycell growth in medium containing a selection agent for the selectablemarker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin,or G-418. See U.S. Pat. No. 4,965,199.

These vectors may comprise an enhancer sequence that facilitatestranscription of a DNA encoding the antibody. Many enhancer sequencesare known from mammalian genes (for example, globin, elastase, albumin,alpha-fetoprotein, and insulin). A frequently used enhancer is onederived from a eukaryotic cell virus. Examples thereof include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers (See,Yaniv, Nature, 297:17-18, 1982, on enhancing elements for activation ofeukaryotic promoters). The enhancer may be spliced into the vector at aposition 5′ or 3′ to the antibody-encoding sequence, but is preferablylocated at a site 5′ from the promoter.

Expression and cloning vectors will also generally comprise a promoterthat is recognized by the host organism and is operably linked to theantibody nucleic acid. Promoter sequences are known for eukaryotes.Virtually all eukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), BPV, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus, and most preferably SV40, from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter, fromheat-shock promoters, provided such promoters are compatible with thehost cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using the BPVas a vector is disclosed in U.S. Pat. No. 4,419,446. A modification ofthis system is described in U.S. Pat. No. 4,601,978. See also Reyes etal., Nature, 297:598-601 (1982) on expression of human beta-interferoncDNA in mouse cells under the control of a thymidine kinase promoterfrom herpes simplex virus. Alternatively, the rous sarcoma virus longterminal repeat can be used as the promoter.

Strong transcription promoters can be used, such as promoters from SV40,cytomegalovirus, or myeloproliferative sarcoma virus. See, e.g., U.S.Pat. No. 4,956,288 and U.S. Patent Publication No. 20030103986. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.Expression vectors for use in mammalian cells include pZP-1, pZP-9, andpZMP21, which have been deposited with the American Type CultureCollection, 10801 University Blvd., Manassas, Va. USA under accessionnumbers 98669, 98668, and PTA-5266, respectively, and derivatives ofthese vectors.

Expression vectors used in eukaryotic host cells (yeast, fungus, insect,plant, animal, human, or a nucleated cell from other multicellularorganism) will also generally contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO 94/11026 and the expression vectordisclosed therein.

Suitable host cells for cloning or expressing the subject antibodiesinclude prokaryote, yeast, or higher eukaryote cells described above.However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture has become a routine procedure. Examplesof useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-1 (ATCC No. CRL 1650); and COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, (ATCC No. CRL 1573; Graham et al., J. Gen.Virol., 36:59-72 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10,ATCC No. CRL 1632; BHK 570, ATCC No. CRL 10314); CHO cells (CHO-K1, ATCCNo. CCL 61; CHO-DG44, Urlaub et al., Proc. Natl. Acad. Sci. USA,77:4216-4220 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2). Additional suitable cell lines are known in the art andavailable from public depositories such as the American Type CultureCollection, Manassas, Va.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences asdiscussed supra.

The mammalian host cells used to produce the antibody of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma-Aldrich Corporation, St. Louis, Mo.), MinimalEssential Medium ((“MEM” (Sigma-Aldrich Corporation, St. Louis, Mo.),Roswell Park Memorial Institute-1640 medium (“RPMI-1640”, Sigma-AldrichCorporation, St. Louis, Mo.), and Dulbecco's Modified Eagle's Medium((“DMEM” Sigma-Aldrich Corporation, St. Louis, Mo.) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enz., 58:44 (1979), Barnes et al., Anal. Biochem., 102:255(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Reexam No. 30,985 canbe used as culture media for the host cells. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas Gentamycin drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan. Methods of development and optimization of media andculture conditions are known in the art (See, Gronemeyer et al.,Bioengineering, 1(4):188-212, 2014).

After culture conditions are optimized and a preferred cell line cloneis selected, these cells are cultured (either adherent cells orsuspension cultures) most typically in a batch-fed process in abioreactor (many models are commercially available) that involvescontinuously feeding the cell culture with medium and feed, optimizedfor the particular cell line chosen and selected for this purpose. (See,Butler, M., Appl. Microbiol. Biotechnol., 68:283-291, 2005; and Kelley,B., mAb, 1(5):443-452, 2009). Perfusion systems are also available inwhich media and feed are continuously supplied to the culture while thesame volume of media is being withdrawn from the bioreactor. (Wurm,2004). Synthetic media, also commercially available, are available forgrowing cells in a batch-fed culture, avoiding the possibility ofcontamination from outside sources, such as with the use of animalcomponents, such as bovine serum albumin, etc. However,animal-component-free hydrolysates are commercially available to helpboost cell density, culture viability and productivity. (Li et al.,2010). Many studies have been performed in an effort to optimize cellculture media, including careful attention to head space available inroller bottles, redox potentials during growth and expression phases,presence of reducing agents to maintain disulfide bonds duringproduction, etc. (See, for instance, Hutterer et al., mAbs,5(4):608-613, 2013; and Mullan et al., BMC Proceed., 5(Suppl 8):P110,2011). Various methodologies have been developed to address thepossibility of harmful oxidation during recombinant monoclonal antibodyproduction. (See, for example, U.S. Pat. No. 8,574,869). Cultured cellsmay be grown by feeding nutrients continuously or as separatelyadministered amounts. Often various process parameters such as cellconcentration, pH, temperature, CO₂, dO₂, osmolality, amount ofmetabolites such as glucose, lactate, glutamine and glutamate, and thelike, are monitored by the use of probes during the cell growth eitheron-line by direct connection to calibrated analyzers or off-line byintervention of operators. The culturing step also typically involvesensuring that the cells growing in culture maintain the transfectedrecombinant genes by any means known in the art for cell selection.

Following fermentation, i.e., upon reaching maximum cell growth andrecombinant protein expression, the culturing step is typically followedby a harvesting step, whereby the cells are separated from the mediumand a harvested cell culture media is thereby obtained. (See, Liu etal., mAbs, 2(5):480-499, 2010). Typically various purification steps,involving column chromatography and the like, follow culturing toseparate the recombinant monoclonal antibody from cell components andcell culture media components. The exact purification steps needed forthis phase of the production of recombinant monoclonal antibodiesdepends on the site of expression of the proteins, i.e., in the cytosolof the cells themselves, or the more commonly preferred route of proteinexcreted into the cell culture medium. Various cell components may beseparated using techniques known in the art such as differentialcentrifugation techniques, gravity-based cell settling, and/or sizeexclusion chromatograph/filtration techniques that can includetangential flow micro-filtration or depth filtration. (See, Pollock etal., Biotechnol. Bioeng., 110:206-219, 2013, and Liu et al., 2010).Centrifugation of cell components may be achieved on a large scale byuse of continuous disk stack centrifuges followed by clarification usingdepth and membrane filters. (See, Kelley, 2009). Most often, afterclarification, the recombinant protein is further purified by Protein Achromatography due to the high affinity of Protein A for the Fc domainof antibodies, and typically occurs using a low pH/acidification elutionstep (typically the acidification step is combined with a precautionaryvirus inactivation step). Flocculation and/or precipitation steps usingacidic or cationic polyelectrolytes may also be employed to separateanimal cells in suspension cultures from soluble proteins. (Liu et al.,2010). Lastly, anion- and cation-exchange chromatography, hydrophobicinteraction chromatograph (“HIC”), hydrophobic charge inductionchromatograph (HCIC), hydroxyapatite chromatography using ceramichydroxyapatite (Ca₅(PO₄)₃OH)₂, and combinations of these techniques aretypically used to polish the solution of recombinant monoclonalantibody. Final formulation and concentration of the desired monoclonalantibody may be achieved by use of ultracentrifugation techniques.Purification yields are typically 70 to 80%. (Kelley, 2009).

The terms “desired protein” or “desired antibody” are usedinterchangeably and refer generally to a parent antibody specific to atarget, i.e., PACAP or a chimeric or humanized antibody or a bindingportion thereof derived therefrom as described herein. The term“antibody” is intended to include any polypeptide chain-containingmolecular structure with a specific shape that fits to and recognizes anepitope, where one or more non-covalent binding interactions stabilizethe complex between the molecular structure and the epitope. Thearchetypal antibody molecule is the immunoglobulin, and all types ofimmunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g.human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken,other avians, etc., are considered to be “antibodies.” A preferredsource for producing antibodies useful as starting material according tothe invention is rabbits. Examples thereof include chimeric antibodies,human antibodies and other non-human mammalian antibodies, humanizedantibodies, single chain antibodies (such as scFvs), camelbodies,nanobodies, IgNAR (single-chain antibodies which may be derived fromsharks, for example), small-modular immunopharmaceuticals (“SMIPs”), andantibody fragments such as Fabs, Fab′, F(ab′)₂, and the like (SeeStreltsov et al., Protein Sci., 14(11):2901-9 (2005); Greenberg et al.,Nature, 374(6518):168-73 (1995); Nuttall et al., Mol. Immunol.,38(4):313-26 (2001); Hamers-Casterman et al., Nature, 363(6428):446-8(1993); Gill et al., Curr. Opin. Biotechnol., (6):653-8 (2006)).

For example, antibodies or antigen binding fragments thereof may beproduced by genetic engineering. In this technique, as with othermethods, antibody-producing cells are sensitized to the desired antigenor immunogen. The messenger RNA isolated from antibody producing cellsis used as a template to make cDNA using PCR amplification. A library ofvectors, each containing one heavy chain gene and one light chain generetaining the initial antigen specificity, is produced by insertion ofappropriate sections of the amplified immunoglobulin cDNA into theexpression vectors. A combinatorial library is constructed by combiningthe heavy chain gene library with the light chain gene library. Thisresults in a library of clones that co-express a heavy and light chain(resembling the Fab fragment or antigen binding fragment of an antibodymolecule). The vectors that carry these genes are co-transfected into ahost cell. When antibody gene synthesis is induced in the transfectedhost, the heavy and light chain proteins self-assemble to produce activeantibodies that can be detected by screening with the antigen orimmunogen.

Antibody coding sequences of interest include those encoded by nativesequences, as well as nucleic acids that, by virtue of the degeneracy ofthe genetic code, are not identical in sequence to the disclosed nucleicacids, and variants thereof. Variant polypeptides can include amino acid(“aa”) substitutions, additions, or deletions. The amino acidsubstitutions can be conservative amino acid substitutions orsubstitutions to eliminate non-essential amino acids, such as to alter aglycosylation site, or to minimize misfolding by substitution ordeletion of one or more cysteine residues that are not necessary forfunction. Variants can be designed so as to retain or have enhancedbiological activity of a particular region of the protein (e.g., afunctional domain, catalytic amino acid residues, etc.). Variants alsoinclude fragments of the polypeptides disclosed herein, particularlybiologically active fragments and/or fragments corresponding tofunctional domains. Techniques for in vitro mutagenesis of cloned genesare known. Also included in the subject invention are polypeptides thathave been modified using ordinary molecular biological techniques so asto improve their resistance to proteolytic degradation or to optimizesolubility properties or to render them more suitable as a therapeuticagent.

Chimeric antibodies may be made by recombinant means by combining theV_(L) and V_(H) regions, obtained from antibody producing cells of onespecies with the constant light and heavy chain regions from another.Typically chimeric antibodies utilize rodent or rabbit variable regionsand human constant regions, in order to produce an antibody withpredominantly human domains. The production of such chimeric antibodiesis well known in the art, and may be achieved by standard means (asdescribed, e.g., in U.S. Pat. No. 5,624,659, incorporated herein byreference in its entirety). It is further contemplated that the humanconstant regions of chimeric antibodies of the invention may be selectedfrom IgG1, IgG2, IgG3, and IgG4 constant regions.

Humanized antibodies are engineered to contain even more human-likeimmunoglobulin domains, and incorporate only the complementaritydetermining regions of the animal-derived antibody. This is accomplishedby carefully examining the sequence of the hyper-variable loops of thevariable regions of the monoclonal antibody and fitting them to thestructure of the human antibody chains. Although facially complex, theprocess is straightforward in practice. See, e.g., U.S. Pat. No.6,187,287, incorporated fully herein by reference.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) may be synthesized.“Fragment” or minimal immunoglobulins may be designed utilizingrecombinant immunoglobulin techniques. For instance “Fv” immunoglobulinsfor use in the present invention may be produced by synthesizing a fusedvariable light chain region and a variable heavy chain region.Combinations of antibodies are also of interest, e.g. diabodies, whichcomprise two distinct Fv specificities. In another embodiment of theinvention, small molecule immunopharmaceuticals (“SMIPs”), camelbodies,nanobodies, and IgNAR are encompassed by immunoglobulin fragments.

Immunoglobulins and fragments thereof may be modifiedpost-translationally, e.g. to add effector moieties such as chemicallinkers, detectable moieties, such as fluorescent dyes, enzymes, toxins,substrates, bioluminescent materials, radioactive materials,chemiluminescent moieties, and the like, or specific binding moieties,such as streptavidin, avidin, or biotin, and the like may be utilized inthe methods and compositions of the present invention. Examples ofadditional effector molecules are provided infra.

A polynucleotide sequence “corresponds” to a polypeptide sequence iftranslation of the polynucleotide sequence in accordance with thegenetic code yields the polypeptide sequence (i.e., the polynucleotidesequence “encodes” the polypeptide sequence), one polynucleotidesequence “corresponds” to another polynucleotide sequence if the twosequences encode the same polypeptide sequence.

A “heterologous” region or domain of a DNA construct is an identifiablesegment of DNA within a larger DNA molecule that is not found inassociation with the larger molecule in nature. Thus, when theheterologous region encodes a mammalian gene, the DNA flanking the geneusually does not flank the mammalian genomic DNA in the genome of thesource organism. Another example of a heterologous region is a constructwhere the coding sequence itself is not found in nature (e.g., a cDNAwhere the genomic coding sequence contains introns or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

A “coding sequence” is an in-frame sequence of codons that correspond toor encode a protein or peptide sequence. Two coding sequences correspondto each other if the sequences or their complementary sequences encodethe same amino acid sequences. A coding sequence in association withappropriate regulatory sequences may be transcribed and translated intoa polypeptide. A polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence. A “promotersequence” is a DNA regulatory region capable of initiating transcriptionof a downstream (3′ direction) coding sequence, and typically containadditional sites for binding of regulatory molecules, e.g.,transcription factors, that affect the transcription of the codingsequence. A coding sequence is “under the control” of the promotersequence or “operatively linked” to the promoter when RNA polymerasebinds the promoter sequence in a cell and transcribes the codingsequence into mRNA, which is then in turn translated into the proteinencoded by the coding sequence.

The general structure of antibodies in vertebrates now is wellunderstood. See Edelman, G. M., Ann. N.Y. Acad. Sci., 190:5 (1971).Antibodies consist of two identical light polypeptide chains ofmolecular weight approximately 23,000 daltons (the “light chain”), andtwo identical heavy chains of molecular weight 53,000-70,000 (the “heavychain”). The four chains are joined by disulfide bonds in a “Y”configuration wherein the light chains bracket the heavy chains startingat the mouth of the “Y” configuration. The “branch” portion of the “Y”configuration is designated the F_(ab) region; the stem portion of the“Y” configuration is designated the F_(C) region. The amino acidsequence orientation runs from the N-terminal end at the top of the “Y”configuration to the C-terminal end at the bottom of each chain. TheN-terminal end possesses the variable region having specificity for theantigen that elicited it, and is approximately 100 amino acids inlength, there being slight variations between light and heavy chain andfrom antibody to antibody.

The variable region is linked in each chain to a constant region thatextends the remaining length of the chain and that within a particularclass of antibody does not vary with the specificity of the antibody(i.e., the antigen eliciting it). There are five known major classes ofconstant regions that determine the class of the immunoglobulin molecule(IgG, IgM, IgA, IgD, and IgE corresponding to γ, μ, α, δ, and ε (gamma,mu, alpha, delta, or epsilon) heavy chain constant regions). Theconstant region or class determines subsequent effector function of theantibody, including activation of complement (see Kabat, E. A.,Structural Concepts in Immunology and Immunochemistry, 2nd Ed., p.413-436, New York, N.Y.: Holt, Rinehart, Winston (1976)), and othercellular responses (see Andrews et al., Clinical Immunology, pp. 1-18,W. B. Sanders, Philadelphia, Pa. (1980); Kohl et al., Immunology, 48:187(1983)); while the variable region determines the antigen with which itwill react. Light chains are classified as either κ (kappa) or λ(lambda). Each heavy chain class can be prepared with either kappa orlambda light chain. The light and heavy chains are covalently bonded toeach other, and the “tail” portions of the two heavy chains are bondedto each other by covalent disulfide linkages when the immunoglobulinsare generated either by hybridomas or by B-cells.

The expression “variable region” or “VR” refers to the domains withineach pair of light and heavy chains in an antibody that are involveddirectly in binding the antibody to the antigen. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain (V_(L)) at one end and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain.

The expressions “complementarity determining region,” “hypervariableregion,” or “CDR” refer to one or more of the hyper-variable orcomplementarity determining regions (“CDRs”) found in the variableregions of light or heavy chains of an antibody (See Kabat et al.,Sequences of Proteins of Immunological Interest, 4^(th) ed., Bethesda,MID: U.S. Dept. of Health and Human Services, Public Health Service,National Institutes of Health (1987)). These expressions include thehypervariable regions as defined by Kabat et al., (Sequences of Proteinsof Immunological Interest, NIH Publication No. 91-3242, Bethesda, MID:U.S. Dept. of Health and Human Services, National Institutes of Health(1983)) or the hypervariable loops in 3-dimensional structures ofantibodies (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)). TheCDRs in each chain are held in close proximity by framework regions(“FRs”) and, with the CDRs from the other chain, contribute to theformation of the antigen binding site. Within the CDRs there are selectamino acids that have been described as the selectivity determiningregions (“SDRs”) that represent the critical contact residues used bythe CDR in the antibody-antigen interaction (see Kashmiri et al.,Methods, 36(1):25-34 (2005)).

An “epitope” or “binding site” is an area or region on an antigen towhich an antigen-binding peptide (such as an antibody) specificallybinds. A protein epitope may comprise amino acid residues directlyinvolved in the binding (also called immunodominant component of theepitope) and other amino acid residues, which are not directly involvedin the binding, such as amino acid residues that are effectively blockedby the specifically antigen binding peptide (in other words, the aminoacid residue is within the “footprint” of the specifically antigenbinding peptide). The term epitope herein includes both types of aminoacid binding sites in any particular region of PACAP, i.e., PACAP38 andPACAP27, that specifically binds to an anti-PACAP antibody. PACAP maycomprise a number of different epitopes, which may include, withoutlimitation, (1) linear peptide antigenic determinants, (2)conformational antigenic determinants that consist of one or morenon-contiguous amino acids located near each other in a mature PACAPconformation; and (3) post-translational antigenic determinants thatconsist, either in whole or part, of molecular structures covalentlyattached to a PACAP protein such as carbohydrate groups. In particular,the term “epitope” includes the specific residues in a protein orpeptide, e.g., PACAP, which are involved in the binding of an antibodyto such protein or peptide as determined by known and accepted methodssuch as alanine scanning techniques. Such methods are exemplifiedherein.

The phrase that an antibody (e.g., first antibody) binds “substantially”or “at least partially” the same epitope as another antibody (e.g.,second antibody) means that the epitope binding site for the firstantibody comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or more of the amino acid residues on the antigen that constitutes theepitope binding site of the second antibody. Also, that a first antibodybinds substantially or partially the same or overlapping epitope as asecond antibody means that the first and second antibodies compete inbinding to the antigen, as described above. Thus, the term “binds tosubstantially the same epitope or determinant as” a monoclonal antibodymeans that an antibody “competes” with the antibody.

The phrase “binds to the same or overlapping epitope or determinant as”an antibody of interest means that an antibody “competes” with saidantibody of interest for at least one, (e.g., at least 2, at least 3, atleast 4, at least 5) or all residues on PACAP to which said antibody ofinterest specifically binds. The identification of one or moreantibodies that bind(s) to substantially or essentially the same epitopeas the monoclonal antibodies described herein can be readily determinedusing alanine scanning. Additionally, any one of variety ofimmunological screening assays in which antibody competition can beassessed. A number of such assays are routinely practiced and well knownin the art (see, e.g., U.S. Pat. No. 5,660,827, issued Aug. 26, 1997,which is specifically incorporated herein by reference). It will beunderstood that actually determining the epitope to which an antibodydescribed herein binds is not in any way required to identify anantibody that binds to the same or substantially the same or overlappingepitope as the monoclonal antibody described herein.

For example, where the test antibodies to be examined are obtained fromdifferent source animals, or are even of a different Ig isotype, asimple competition assay may be employed in which the control antibodyis mixed with the test antibody and then applied to a sample containingPACAP. Protocols based upon ELISAs, radioimmunoassays, Western blotting,and the use of BIACORE® (GE Healthcare Life Sciences, Marlborough,Mass.) analysis are suitable for use in such simple competition studies.

In certain embodiments, the control anti-PACAP antibody is pre-mixedwith varying amounts of the test antibody (e.g., in ratios of about 1:1,1:2, 1:10, or about 1:100) for a period of time prior to applying to thePACAP38 or PACAP27 antigen sample. In other embodiments, the control andvarying amounts of test antibody can simply be added separately andadmixed during exposure to the PACAP38 or PACAP27 antigen sample. Aslong as bound antibodies can be distinguished from free antibodies(e.g., by using separation or washing techniques to eliminate unboundantibodies) and control antibody from the test antibody (e.g., by usingspecies specific or isotype specific secondary antibodies or byspecifically labeling the control antibody with a detectable label) itcan be determined if the test antibody reduces the binding of thecontrol antibody to the PACAP38 or PACAP27 antigens, indicating that thetest antibody recognizes substantially the same epitope as the controlanti-PACAP antibody. The binding of the (labeled) control antibody inthe presence of a completely irrelevant antibody (that does not bindPACAP) can serve as the control high value. The control low value can beobtained by incubating the labeled control antibody with the same butunlabeled control antibody, where competition would occur and reducebinding of the labeled antibody. In a test assay, a significantreduction in labeled antibody reactivity in the presence of a testantibody is indicative of a test antibody that recognizes substantiallythe same epitope, i.e., one that competes with the labeled controlantibody. For example, any test antibody that reduces the binding of thecontrol antibody to PACAP38 or PACAP27 by at least about 50%, such as atleast about 60%, or more preferably at least about 70% (e.g., about65-100%), at any ratio of test antibody between about 1:1 or 1:10 andabout 1:100 is considered to be an antibody that binds to substantiallythe same or overlapping epitope or determinant as the control antibody.

Preferably, such test antibody will reduce the binding of the controlantibody to PACAP38 or PACAP27 antigen preferably at least about 50%, atleast about 60%, at least about 80%, or at least about 90% (e.g., about95%) of the binding of the control antibody observed in the absence ofthe test antibody.

A simple competition assay in which a test antibody is applied atsaturating concentration to a surface onto which PACAP38 or PACAP27 isimmobilized also may be advantageously employed. The surface in thesimple competition assay is preferably a BIACORE® (GE Healthcare LifeSciences, Marlborough, Mass.) chip (or other media suitable for surfaceplasmon resonance (“SPR”) analysis). The binding of a control antibodythat binds PACAP38 or PACAP27 to the PACAP-coated surface is measured.This binding to the PACAP38- or PACAP27-containing surface of thecontrol antibody alone is compared with the binding of the controlantibody in the presence of a test antibody. A significant reduction inbinding to the PACAP38- or PACAP27-containing surface by the controlantibody in the presence of a test antibody indicates that the testantibody recognizes substantially the same epitope as the controlantibody such that the test antibody “competes” with the controlantibody. Any test antibody that reduces the binding of control antibodyby at least about 20% or more, at least about 40%, at least about 50%,at least about 70%, or more, can be considered to be an antibody thatbinds to substantially the same epitope or determinant as the controlantibody. Preferably, such test antibody will reduce the binding of thecontrol antibody to PACAP38 or PACAP27 by at least about 50% (e.g., atleast about 60%, at least about 70%, or more). It will be appreciatedthat the order of control and test antibodies can be reversed; i.e. thecontrol antibody can be first bound to the surface and then the testantibody is brought into contact with the surface thereafter in acompetition assay. Preferably, the “sandwich-style” binding assayexemplified in Example 9 infra is used. Alternatively, the antibodyhaving greater affinity for PACAP38 or PACAP27 antigen is bound to thePACAP38- or PACAP27-containing surface first, as it will be expectedthat the decrease in binding seen for the second antibody (assuming theantibodies are competing) will be of greater magnitude. Further examplesof such assays are provided in e.g., Saunal and Regenmortel, J. Immunol.Methods, 183:33-41 (1995), the disclosure of which is incorporatedherein by reference.

In addition, whether an antibody binds the same or overlappingepitope(s) on PACAP as another antibody or the epitope bound by a testantibody may in particular be determined using a Western-blot basedassay. In this assay a library of peptides corresponding to the antigenbound by the antibody, the PACAP protein, is made, that compriseoverlapping portions of the protein, typically 10-25, 10-20, or 10-15amino acids long. These different overlapping amino acid peptidesencompassing the PACAP sequence are synthesized and covalently bound toa PEPSPOTS' nitrocellulose membrane (JPT Peptide Technologies, Berlin,Germany). Blots are then prepared and probed according to themanufacturer's recommendations.

Essentially, the immunoblot assay then detects by fluorometric meanswhat peptides in the library bind to the test antibody and thereby canidentify what residues on the antigen, i.e., PACAP, interact with thetest antibody. (See U.S. Pat. No. 7,935,340, incorporated by referenceherein).

Various epitope mapping techniques are known in the art. By way ofexample, X-ray co-crystallography of the antigen and antibody; NMR; SPR(e.g., at 25° or 37° C.); array-based oligo-peptide scanning (or“pepscan analysis”); site-directed mutagenesis (e.g., alanine scanning);mutagenesis mapping; hydrogen-deuterium exchange; phage display; andlimited proteolysis are all epitope mapping techniques that are wellknown in the art (See, e.g., Epitope Mapping Protocols: Second Edition,Methods in Molecular Biology, editors Mike Schutkowski and UlrichReineke, 2^(nd) Ed., New York, N.Y.: Humana Press (2009), and EpitopeMapping Protocols, Methods in Molecular Biology, editor Glenn Morris,1^(st) Ed., New York, N.Y.: Humana Press (1996), both of which areherein incorporated by referenced in their entirety).

The identification of one or more antibodies that bind(s) tosubstantially or essentially the same epitope as the monoclonalantibodies described herein, e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7,Ab8, Ab9, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, orAb1.H, can be readily determined using any one of variety ofimmunological screening assays in which antibody competition can beassessed. A number of such assays are routinely practiced and well knownin the art (see, e.g., U.S. Pat. No. 5,660,827, issued Aug. 26, 1997,which is incorporated herein by reference). It will be understood thatdetermining the epitope to which an antibody described herein binds isnot in any way required to identify an antibody that binds to the sameor substantially the same epitope as the monoclonal antibody describedherein.

For example, where the test antibodies to be examined are obtained fromdifferent source animals, or are even of a different Ig isotype, asimple competition assay may be employed in which the control antibody(one of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13,Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, or Ab1.H, for example) is mixed withthe test antibody and then applied to a sample containing either or bothPACAP38 and PACAP27, each of which is known to be bound by Ab1, Ab2,Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16,Ab17, Ab18, Ab19, Ab1.H. Protocols based upon ELISAs, radioimmunoassays,Western blotting, and BIACORE® (GE Healthcare Life Sciences,Marlborough, Mass.) analysis (as described in the Examples sectionherein) are suitable for use in such simple competition studies.

In certain embodiments, the method comprises pre-mixing the controlantibody with varying amounts of the test antibody (e.g., in ratios ofabout 1:1, 1:2, 1:10, or about 1:100) for a period of time prior toapplying to the PACAP antigen sample. In other embodiments, the controland varying amounts of test antibody can be added separately and admixedduring exposure to the PACAP antigen sample. As long as bound antibodiescan be distinguished from free antibodies (e.g., by using separation orwashing techniques to eliminate unbound antibodies) and control antibodyfrom the test antibody (e.g., by using species specific or isotypespecific secondary antibodies or by specifically labelling the controlantibody with a detectable label), the method can be used to determinethat the test antibody reduces the binding of the control antibody tothe PACAP antigen, indicating that the test antibody recognizessubstantially the same epitope as the control antibody (e.g., Ab1, Ab2,Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16,Ab17, Ab18, Ab19, or Ab1.H). The binding of the (labeled) controlantibody in the presence of a completely irrelevant antibody (that doesnot bind PACAP) can serve as the control high value. The control lowvalue can be obtained by incubating the labeled control antibody withthe same but unlabeled control antibody, where competition would occurand reduce binding of the labeled antibody. In a test assay, asignificant reduction in labeled antibody reactivity in the presence ofa test antibody is indicative of a test antibody that recognizessubstantially the same epitope, i.e., one that competes with the labeledcontrol antibody. For example, any test antibody that reduces thebinding of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12,Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, or Ab1.H to both of PACAP38and PACAP27 antigens by at least about 50%, such as at least about 60%,or more preferably at least about 70% (e.g., about 65-100%), at anyratio of control Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11,Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, or Ab1.H:test antibodyor Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13, Ab14,Ab15, Ab16, Ab17, Ab18, Ab19, or Ab1.H:test antibody between about 1:1or 1:10 and about 1:100 is considered to be an antibody that binds tosubstantially the same epitope or determinant as Ab1, Ab2, Ab3, Ab4,Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18,Ab19, or Ab1.H, respectively. Preferably, such test antibody will reducethe binding of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12,Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, or Ab1.H to at least one,preferably each, of the PACAP38 and PACAP27 antigens preferably at leastabout 50%, at least about 60%, at least about 80% or at least about 90%(e.g., about 95%) of the binding of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7,Ab8, Ab9, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, or Ab1.Hobserved in the absence of the test antibody. These methods can beadapted to identify and/or evaluate antibodies that compete with othercontrol antibodies.

A simple competition assay in which a test antibody is applied atsaturating concentration to a surface onto which either PACAP38 orPACAP27, or both, are immobilized also may be advantageously employed.The surface in the simple competition assay is preferably of a mediasuitable for OCTET® and/or PROTEON®. The binding of a control antibody(e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13,Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, or Ab1.H) to the PACAP-coatedsurface is measured. This binding to the PACAP-containing surface of thecontrol antibody alone is compared with the binding of the controlantibody in the presence of a test antibody. A significant reduction inbinding to the PACAP-containing surface by the control antibody in thepresence of a test antibody indicates that the test antibody recognizessubstantially the same epitope as the control antibody such that thetest antibody “competes” with the control antibody. Any test antibodythat reduces the binding of control antibody (such as Ab1, Ab2, Ab3,Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17,Ab18, Ab19, or Ab1.H) to both of PACAP38 and PACAP27 antigens by atleast about 20% or more, at least about 40%, at least about 50%, atleast about 70%, or more, can be considered to be an antibody that bindsto substantially the same epitope or determinant as the control antibody(e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13,Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, or Ab1.H). Preferably, such testantibody will reduce the binding of the control antibody (e.g., Ab1,Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13, Ab14, Ab15,Ab16, Ab17, Ab18, Ab19, or Ab1.H) to the PACAP antigen by at least about50% (e.g., at least about 60%, at least about 70%, or more). It will beappreciated that the order of control and test antibodies can bereversed; i.e. the control antibody can be first bound to the surfaceand then the test antibody is brought into contact with the surfacethereafter in a competition assay. Preferably, the antibody havinghigher affinity for PACAP38 and PACAP27 is bound to the PACAP-containingsurface first, as it will be expected that the decrease in binding seenfor the second antibody (assuming the antibodies are competing) will beof greater magnitude. Further examples of such assays are provided in,e.g., Saunal and Regenmortel, J. Immunol. Methods, 183:33-41 (1989), thedisclosure of which is incorporated herein by reference.

Determination of whether an antibody, antigen binding fragment thereof,or antibody derivative binds within one of the epitope regions definedabove can be carried out in ways known to the person skilled in the art.In another example of such mapping/characterization methods, an epitoperegion for an anti-PACAP antibody may be determined by epitope“footprinting” using chemical modification of the exposedamines/carboxyls in the PACAP38 and PACAP27 protein. One specificexample of such a foot-printing technique is the use ofhydrogen-deuterium exchange detected by mass spectrometry (“HXMS”),wherein a hydrogen/deuterium exchange of receptor and ligand proteinamide protons, binding, and back exchange occurs, wherein the backboneamide groups participating in protein binding are protected from backexchange and therefore will remain deuterated. Relevant regions can beidentified at this point by peptic proteolysis, fast microborehigh-performance liquid chromatography separation, and/or electrosprayionization mass spectrometry (See, e.g., Ehring H., AnalyticalBiochemistry, 267(2):252-259 (1999) and Engen, J. R. & Smith, D. L.,Anal. Chem., 73:256A-265A (2001)). Another example of a suitable epitopeidentification technique is nuclear magnetic resonance epitope mapping(“NMR”), where typically the position of the signals in two-dimensionalNMR spectres of the free antigen and the antigen complexed with theantigen binding peptide, such as an antibody, are compared. The antigentypically is selectively isotopically labeled with ¹⁵N so that onlysignals corresponding to the antigen and no signals from the antigenbinding peptide are seen in the NMR-spectrum. Antigen signalsoriginating from amino acids involved in the interaction with theantigen binding peptide typically will shift position in the spectres ofthe complex compared to the spectres of the free antigen, and the aminoacids involved in the binding can be identified that way, See, e.g.,Ernst Schering Res. Found. Workshop, (44):149-67 (2004); Huang et al.,J. Mol. Biol., 281(1):61-67 (1998); and Saito and Patterson, Methods,9(3):516-24 (1996)). Epitope mapping/characterization also can beperformed using mass spectrometry (“MS”) methods (See, e.g., Downard, J.Mass Spectrom., 35(4):493-503 (2000) and Kiselar and Downard, Anal.Chem., 71(9):1792-801 (1999)).

Protease digestion techniques also can be useful in the context ofepitope mapping and identification. Antigenic determinant-relevantregions/sequences can be determined by protease digestion, e.g. by usingtrypsin in a ratio of about 1:50 to PACAP38 or PACAP27 overnight (“o/n”)digestion at 37° C. and pH 7-8, followed by mass spectrometry (“MS”)analysis for peptide identification. The peptides protected from trypsincleavage by the anti-PACAP antibody can subsequently be identified bycomparison of samples subjected to trypsin digestion and samplesincubated with antibody and then subjected to digestion by e.g. trypsin(thereby revealing a footprint for the antibody). Other enzymes likechymotrypsin or pepsin can be used in similar epitope characterizationmethods. Moreover, enzymatic digestion can provide a quick method foranalyzing whether a potential antigenic determinant sequence is within aregion of PACAP in the context of a PACAP-binding polypeptide. If thepolypeptide is not surface exposed, it is most likely not relevant interms of immunogenicity/antigenicity (See, e.g., Manca, Ann. Ist. Super.Sanità, 27(1):15-9 (1991) for a discussion of similar techniques).

Site-directed mutagenesis is another technique useful forcharacterization of a binding epitope. For example, in“alanine-scanning” site-directed mutagenesis (also known as alaninescanning, alanine scanning mutagenesis, alanine scanning mutations,combinatorial alanine scanning, or creation of alanine point mutations,for example), each residue within a protein segment is replaced with analanine residue (or another residue such as valine where alanine ispresent in the wild-type sequence) through such methodologies as directpeptide or protein synthesis, site-directed mutagenesis, the GENEART™Mutagenesis Service (Thermo Fisher Scientific, Waltham, Mass. U.S.A.) orshotgun mutagenesis, for example. A series of single point mutants ofthe molecule is thereby generated using this technique; the number ofmutants generated is equivalent to the number of residues in themolecule, each residue being replaced, one at a time, by a singlealanine residue. Alanine is generally used to replace native (wild-type)residues because of its non-bulky, chemically inert, methyl functionalgroup that can mimic the secondary structure preferences that many otheramino acids may possess. Subsequently, the effects replacing a nativeresidue with an alanine has on binding affinity of an alanine scanningmutant and its binding partner can be measured using such methods as,but not limited to, SPR binding experiments. If a mutation leads to asignificant reduction in binding affinity, it is most likely that themutated residue is involved in binding. Monoclonal antibodies specificfor structural epitopes (i.e., antibodies that do not bind the unfoldedprotein) can be used as a positive control for binding affinityexperiments to verify that the alanine-replacement does not influencethe overall tertiary structure of the protein (as changes to the overallfold of the protein may indirectly affect binding and thereby produce afalse positive result). See, e.g., Clackson and Wells, Science,267:383-386 (1995); Weiss et al., Proc. Natl. Acad. Sci. USA,97(16):8950-8954 (2000); and Wells, Proc. Natl. Acad. Sci. USA, 93:1-6(1996). In Example 12 alanine scanning methods are used to identify thespecific epitope or residues of PACAP which specifically interact withthe anti-PACAP antibodies disclosed herein.

Electron microscopy can also be used for epitope “footprinting”. Forexample, Wang et al., Nature, 355:275-278 (1992) used coordinatedapplication of cryoelectron microscopy, three-dimensional imagereconstruction, and X-ray crystallography to determine the physicalfootprint of a Fab-fragment on the capsid surface of native cowpeamosaic virus.

Other forms of “label-free” assay for epitope evaluation include SPR(sold commercially as the BIACORE® system, GE Healthcare Life Sciences,Marlborough, Mass.) and reflectometric interference spectroscopy(“RifS”) (See, e.g., Fagerstam et al., Journal of Molecular Recognition,3:208-14 (1990); Nice et al., J. Chromatogr., 646:159-168 (1993);Leipert et al., Angew. Chem. Int. Ed., 37:3308-3311 (1998); Kroger etal., Biosensors and Bioelectronics, 17:937-944 (2002)).

The expressions “framework region” or “FR” refer to one or more of theframework regions within the variable regions of the light and heavychains of an antibody (See Kabat et al., Sequences of Proteins ofImmunological Interest, 4^(th) edition, Bethesda, Md.: U.S. Dept. ofHealth and Human Services, Public Health Service, National Institutes ofHealth (1987)). These expressions include those amino acid sequenceregions interposed between the CDRs within the variable regions of thelight and heavy chains of an antibody.

The term “Fc region” is used to define a C-terminal region of animmunoglobulin heavy chain. The “Fc region” may be a native sequence Fcregion or a variant Fc region. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy chainFc region is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. Thenumbering of the residues in the Fc region is that of the EU index as inKabat. Kabat et al., Sequences of Proteins of Immunological Interest,5th edition, Bethesda, Md.: U.S. Dept. of Health and Human Services,Public Health Service, National Institutes of Health (1991). The Fcregion of an immunoglobulin generally comprises two constant domains,CH2 and CH3.

The terms “Fc receptor” and “FcR” describe a receptor that binds to theFc region of an antibody. The preferred FcR is a native sequence humanFcR. Moreover, a preferred FcR is one that binds an IgG antibody (agamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. FcRs are reviewed in Ravetch and Kinet, Ann. Rev. Immunol.,9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and deHaas et al., J. Lab. Clin. Med., 126:330-41 (1995). “FcR” also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol., 117:587 (1976);and Kim et al., J. Immunol., 24:249 (1994)), and which primarilyfunctions to modulate and/or extend the half-life of antibodies incirculation. To the extent that the disclosed anti-PACAP antibodies areaglycosylated, as a result of the expression system and/or sequence, thesubject antibodies are expected to bind FcRn receptors, but not to bind(or to minimally bind) Fcγ receptors.

A “functional Fc region” possesses at least one effector function of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity (“CDC”); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (“ADCC”); phagocytosis;down-regulation of cell surface receptors (e.g. B cell receptor(“BCR”)), etc. Such effector functions generally require the Fc regionto be combined with a binding domain (e.g. an antibody variable domain)and can be assessed using various assays known in the art for evaluatingsuch antibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence that differs from that of anative sequence Fc region by virtue of at least one amino acidmodification, yet retains at least one effector function of the nativesequence Fc region. Preferably, the variant Fc region has at least oneamino acid substitution compared to a native sequence Fc region or tothe Fc region of a parent polypeptide, e.g. from about one to about tenamino acid substitutions, and preferably from about one to about fiveamino acid substitutions in a native sequence Fc region or in the Fcregion of the parent polypeptide. The variant Fc region herein willpreferably possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, andmost preferably at least about 90% sequence identity therewith, morepreferably at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99% sequence identity therewith.Anti-PACAP Antibodies and Binding Fragments Thereof Having BindingAffinity for PACAP

PACAP is a multifunctional vasodilatory peptide with expressionthroughout the central nervous system (“CNS”) and periphery. PACAP is amember of the secretin/VIP/GRH family. PACAP exists in two α-amidatedactive forms, PACAP38 (SEQ ID NO: 1241) and PACAP27 (SEQ ID NO: 1242).Herein, the term “PACAP” includes either or both of PACAP38 and PACAP27unless expressly indicated otherwise. PACAP is highly conserved betweenspecies.

In humans, PACAP is derived from a 176 amino acid precursor protein(preproPACAP) and the gene is located on chromosome 18p11, with PACAP38encoded for by exon 5 (Vaudry et al., Pharmacol. Rev., 61:283-357(2009)). PreproPACAP contains an N-terminal 24 amino acid signalprotein, a 29 amino acid PACAP-related peptide and PACAP in theC-terminal domain. The precursor is metabolized by prohormone convertaseenzymes into biologically active PACAP38 and PACAP27.

VIP (SEQ ID NO: 1243) belongs to the same protein family as PACAP andshares high homology with PACAP, i.e., VIP and PACAP27 have 68% sequencehomology at the amino acid level, as well as similar overall secondarystructure, i.e. long alpha-helical structures at the C-terminus.

PACAP's actions are mediated via three different G-protein coupledreceptors: PAC1-R, VPAC1-R, and VPAC2-R. VPAC1-R can associate with allof the receptor-associated membrane proteins (“RAMPs”, Kaiser & Russo,Neuropeptides 47: 451-461 (2013)). PAC1-R is selective for PACAP,whereas VPAC1-R and VPAC2-R bind to both VIP and PACAP with highaffinity. PAC1-R binds to PACAP with 100-1000-fold higher affinity thanVIP, i.e., K_(D)-0.5 nM for PACAP27/PACAP38 vs. K_(D)˜500 nM for VIP.Conversely, VPAC1-R and VPAC2-R have equal affinities for PACAP and VIP(K_(D)˜1 nM) (See Schytz et al. (2010)). All three receptors are widelyexpressed in both peripheral tissues and in the CNS, with PAC1-Rpredominantly expressed in the CNS, most abundantly in the olfactorybulb, thalamus, hypothalamus, the dentate gyrus of the hippocampus andin granule cells of the cerebellum (Hashimoto et al., J. Comp. Neurol.,371:567-577 (1996); Shioda et al., Neurosci. Res., 28:345-354 (1997)).

Activation of the PAC1-R, VPAC1-R, and/or VPAC2-R results in increasedadenylate cyclase activity and, thus, increased cAMP production.However, PACAP receptors can also mediate their effects through PLC,leading to increased Ca²⁺ levels, and PLD.

PACAP has a wide range of biological effects, including a role inneurodevelopment, neuroprotection, neuromodulation, neurogenicinflammation, and nociception. PACAP is also reported to interact withglycosaminoglycans (“GAGs”). GAGs are long, unbranched polysaccharidescomposed of repeating disaccharide units, such as heparin, chondroitin,keratin, and hyaluronic acid. It has been shown that the cellular uptakeof PACAP is dependent on the expression of GAG proteins and that PACAPbound to sulfated GAGs. Particularly, it was determined that PACAP38binding to GAGs was capable of inducing receptor-independent cellularuptake of PACAP38. This study further demonstrated that a randomcoil-to-α-helix transition in PACAP38 was essential for GAG-dependentuptake of PACAP38, as a mutant PACAP38 that could not undergo thestructural transition was not internalized by GAG-containing cell linesas efficiently as the wild-type form of PACAP38 (Neree et al., FEBSLett., 588(24):4590-4596, 2014). In a follow up study, it was determinedthat PACAP's ability to cluster GAGs, i.e., heparin, was directlyrelated to its ability to function as a cell penetrating peptide(“CPP”). It is hypothesized that this activity is attributable to theheparin-binding, or Cardin-Weintraub, motif found insecretin/glucagon/GHRH family members, such as PACAP (Neree et al., Int.J. Mol. Sci., 16:27391-27400, 2015). Interestingly, Neree et al. (2015)presented data demonstrating that PACAP38 was able to cluster sulfatedGAGs in vitro. These data suggested that the observed clustering effectis important for the GAG-mediated cellular uptake of PACAP38, as otherpeptides, such as glucagon, displayed higher binding affinities forsulfated GAGs (heparin) but are not internalized by cells as efficientlyas PACAP38. Further, it is reported that in in vitro studies in whichcells are exposed to PACAP, cartilage formation is increased, includingcartilage matrix that is rich in sulphated GAG proteins, consistent withits putative protective role expressed during various cellular stressresponses (Juhász et al., PLoS ONE, 9(3):e91541, 2014). Using cell typesthat lack PACAP-specific receptors on their plasma membranes, such asCHO-K1 cells, Doan et al. presented data demonstrating the ability ofsuch cells to engage in receptor-independent cellular uptake of variousforms of fluorescently-labeled PACAP38 and PACAP27 (Doan et al.,Biochem. Biophys. Acta, 1823:940-949, 2012).

The present invention provides exemplary antibodies or antigen bindingfragments thereof that bind PACAP, including human PACAP. Otherantibodies or antigen binding fragments thereof that bind PACAP,including those having different CDRs, and epitopic specificity may beobtained using the disclosure of the present specification, and usingmethods that are generally known in the art. Such antibodies and antigenbinding fragments thereof antagonize the biological effects of PACAP invivo and therefore are useful in treating or preventing PACAP-relatedconditions including, for example, headache, migraine, pain,photophobia, hot flush, PTSD, and anxiety disorders. In preferredembodiments, the antibody or antigen binding fragment thereof accordingto the invention comprises one or more CDRs, a V_(L) chain and/or V_(H)chain of the anti-PACAP antibodies and antigen binding fragments thereofdescribed herein.

In some embodiments, an anti-PACAP antibody or antigen binding fragmentthereof according to the invention will interfere with, block, reduce,or modulate the interaction between PACAP and its receptor(s) (e.g.,PAC1-R, VPAC1-R, and VPAC2-R). In some instances an anti-PACAP antibodyor antigen binding fragment thereof according to the invention is“neutralizing”, e.g., it totally prevents the specific interaction ofPACAP with PAC1-R, VPAC1-R, and/or VPAC2-R. In some embodiments, theantibody or antigen binding fragment thereof neutralizes PACAP, e.g., byremaining bound to PACAP in a location and/or manner that prevents PACAPfrom specifically binding to PAC1-R, VPAC1-R, and/or VPAC2-R.

In some embodiments, the antibody or antigen binding fragment thereofaccording to the invention is capable of inhibiting PACAP-mediatedactivity (including binding to PAC1-R-expressing cells). In someembodiments, the antibody or antigen binding fragment thereof accordingto the invention are humanized, such as humanized rabbit antibodies toPACAP.

As mentioned, the anti-PACAP antibodies or antigen binding fragmentsthereof according to the invention have a variety of uses. For example,the subject antibodies and fragments can be useful in therapeuticapplications, as well as diagnostically in binding assays. The subjectanti-PACAP antibodies or antigen binding fragments thereof are usefulfor affinity purification of PACAP, in particular human PACAP or itsligands and in screening assays to identify other antagonists of PACAPactivity. Some of the antibodies or antigen binding fragments thereofare useful for inhibiting binding of PACAP to PAC1-R, VPAC1-R, and/orVPAC2-R, or inhibiting PACAP-mediated activities and/or biologicaleffects.

As used herein, the term “one or more biological effects associated withPACAP refers to any biological effect mediated, induced, or otherwiseattributable to PACAP, e.g., binding properties, functional properties,and other properties of biological significance. Non-limiting exemplarybiological effects of PACAP include PACAP binding to PAC1-R, VPAC1-R,GAGs, and/or VPAC2-R; PACAP activating PAC1-R, VPAC1-R, and/orVPAC2-R-mediated signaling; PACAP-mediated increase in cAMP production;PACAP-mediated increase in PLC activity; PACAP-mediated increase in PLDactivity; PACAP-mediated increase in Ca²⁺ levels; and PACAP-mediatedvasodilation, photophobia, mast cell degranulation, and/or neuronalactivation. The subject anti-PACAP antibodies are capable of inhibitingone, a combination of, or all of these exemplary PACAP biologicalactivities. For example, the anti-PACAP antibodies and antigen bindingfragments thereof provided herein are capable of inhibitingPACAP-induced vasodilation (see Example 7 and Example 8).

The antibody or antigen binding fragment thereof according to theinvention can be used in a variety of therapeutic applications. Forexample, in some embodiments the anti-PACAP antibody or antigen bindingfragment thereof are useful for treating conditions associated withPACAP, such as, but not limited to, migraine (with or without aura),hemiplegic migraines, cluster headaches, migrainous neuralgia, chronicheadaches, tension headaches, general headaches, hot flush, photophobia,chronic paroxysmal hemicrania, secondary headaches due to an underlyingstructural problem in the head or neck, cranial neuralgia, sinusheadaches (e.g., headache associated with sinusitis), allergy-inducedheadaches or migraines, pain, chronic pain, neuroinflammatory orinflammatory pain, post-operative incision pain, post-surgical pain,trauma-related pain, eye pain, tooth pain, complex regional painsyndrome, cancer pain (e.g., primary or metastatic bone cancer pain),fracture pain, osteoporotic fracture pain, pain resulting from burn,gout joint pain, pain associated with sickle cell crises, painassociated with temporomandibular disorders, cirrhosis, hepatitis,neurogenic pain, neuropathic pain, nociceptic pain, visceral pain,trigeminal neuralgia, post-herpetic neuralgia, phantom limb pain,fibromyalgia, menstrual pain, ovarialgia, reflex sympathetic dystrophy,osteoarthritis or rheumatoid arthritis pain, lower back pain, diabeticneuropathy, sciatica, dyspepsia, irritable bowel syndrome, inflammatorybowel disease, Crohn's disease, ileitis, ulcerative colitis, renalcolic, dysmenorrhea, cystitis, interstitial cystitis, menstrual period,labor, menopause, pancreatitis, schizophrenia, depression, PTSD, anxietydisorders, diabetes, autoimmune diabetes, endothelial dysfunction,ischemia, Raynaud's syndrome, coronary heart disease (“CHD”), coronaryartery disease (“CAD”), heart failure, peripheral arterial disease(“PAD”), pulmonary hypertension (“PH”), connective tissue disorders,stroke, Sjögren's syndrome, multiple sclerosis, bronchialhyperreactivity, asthma, bronchitis, bronchodilation, emphysema, chronicobstructive pulmonary disease (“COPD”), inflammatory dermatitis,adenocarcinoma in glandular tissue, blastoma in embryonic tissue oforgans, carcinoma in epithelial tissue, leukemia in tissues that formblood cells, lymphoma in lymphatic tissue, myeloma in bone marrow,sarcoma in connective or supportive tissue, adrenal cancer, AIDS-relatedlymphoma, anemia, bladder cancer, bone cancer, brain cancer, breastcancer, carcinoid tumors, cervical cancer, chemotherapy, colon cancer,cytopenia, endometrial cancer, esophageal cancer, gastric cancer, headcancer, neck cancer, hepatobiliary cancer, kidney cancer, leukemia,liver cancer, lung cancer, lymphoma, Hodgkin's disease, non-Hodgkin's,nervous system tumors, oral cancer, ovarian cancer, pancreatic cancer,prostate cancer, rectal cancer, skin cancer, stomach cancer, testicularcancer, thyroid cancer, urethral cancer, cancer of bone marrow, multiplemyeloma, tumors that metastasize to the bone, tumors infiltrating thenerve and hollow viscus, tumors near neural structures, acne vulgaris,atopic dermatitis, urticaria, keloids, hypertrophic scars and rosacea,allergic dermatitis, psoriasis, pruritus, neurogenic cutaneous redness,erythema, weight loss, anorexia, sarcoidosis, shock, sepsis, opiatewithdrawal syndrome, morphine tolerance, epilepsy, LUT disorders such asurinary tract infection, abnormal voiding, urinary urgency, nocturia,urinary incontinence, overactive bladder, and for preventing oralleviating the pain associated with such LUT conditions.

Specific examples of visceral pain, i.e., pain associated with theviscera, or the internal organs of the body include pain that affectsorgans such as e.g., the heart, lungs, reproductive organs, bladder,ureters, the digestive organs, liver, pancreas, spleen, and kidneys.Conditions associated therewith include by way of example pancreatitis,labor, abdominal surgery associated with ileus, cystitis, menstrualperiod, or dysmenorrhea. Likewise, kidney pain, epigastric pain, pleuralpain, and painful biliary colic, appendicitis pain may all be consideredto be visceral pain. Substernal pain or pressure from early myocardialinfarction is also visceral. Diseases of the stomach, duodenum or coloncan cause visceral pain. Commonly encountered gastrointestinal (“GI”)disorders that cause visceral pain include functional bowel disorder(“FBD”) and inflammatory bowel disease (“IBD”). Such GI disorders mayfurther include gastro-esophageal reflux, dyspepsia, irritable bowelsyndrome (“IBS”) and functional abdominal pain syndrome (“FAPS”), and,with respect to IBD, Crohn's disease, ileitis, and ulcerative colitis.

The subject anti-PACAP antibodies and antigen binding fragments thereofmay be used alone or in association with other active agents or drugs,including other biologics, to treat any subject in which blocking,inhibiting, or neutralizing the in vivo effect of PACAP or blocking orinhibiting the interaction of PACAP and its receptors, PAC1-R, VPAC1-R,and VPAC2-R, is therapeutically desirable.

Exemplary anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, and the specific CDRs thereof are identifiedin this section. For convenience, each exemplified antibody or antigenbinding fragment thereof, and corresponding sequences are separatelyidentified by a specific nomenclature, i.e., Ab1, Ab1.H, Ab2, Ab3, Ab4,Ab5, Ab6, Ab7, Ab8, Ab9, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18,and Ab19,

The anti-PACAP antibodies and antigen binding fragments thereofcomprising the invention have binding affinity for PACAP, wherein thebinding affinity comprises anti-PACAP antibodies or antigen bindingfragments thereof specifically binding to PACAP38 and PACAP27, but notbinding VIP, and/or antibodies or antigen binding fragments thereofspecifically binding to PACAP38, but not binding to PACAP27 or VIP,and/or antibodies or antigen binding fragments thereof specificallybinding to a linear and/or conformational epitope within PACAP38 and/orPACAP27. More specifically, the epitopes of PACAP38 and/or PACAP27 towhich antagonistic anti-PACAP antibodies or antigen binding fragmentsthereof according to the invention bind will include those which areidentified in Example 12 or residues thereof (as determined by use ofalanine scanning) and/or other epitopic identification methods.

Anti-PACAP Antibody Polypeptides and Polynucleotides Encoding thePolypeptides Antibody Ab1.H

In one embodiment, the invention includes the use or administration ofantibodies and antigen binding fragments having binding specificity toPACAP that possess a heavy chain sequence comprising the sequence of SEQID NO: 41 which consists of the heavy chain variable region of SEQ IDNO: 42 linked to the heavy chain constant region of SEQ ID NO: 50.

In one embodiment, the invention includes the use or administration ofantibodies and antigen binding fragments having binding specificity toPACAP that contain a variable heavy chain sequence comprising thesequence set forth below:

(SEQ ID NO: 42) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSAVMNWVRQAPGKGLEWIGSIVASGTTYYASSANGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RGGGEFFIWGQGTLVTVSS.

In another embodiment, the invention includes the use of antibodies andantigen binding fragments thereof having binding specificity to PACAPthat possess the same epitopic specificity as Ab1.H, and which contain aconstant heavy chain sequence comprising the polypeptide of SEQ ID NO:1244, 1245, or 1246, or comprising the sequence set forth below:

(SEQ ID NO: 50) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes the use or administrationof antibodies and antigen binding fragments having binding specificityto PACAP that contain a light chain sequence comprising the sequence ofSEQ ID NO: 61 which consists of the light chain variable region of SEQID NO: 62 linked to the light chain constant region of SEQ ID NO: 70.

In another embodiment, the invention includes the use or administrationof antibodies and antigen binding fragments having binding specificityto PACAP that contain a variable light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 62) DIQMTQSPSTLSASVGDRVTITCQSSESVYSNYLSWYQQKPGKAPKFLIYQASNLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCAGGYSEN IVGFGGGTKVEIKR.

In another embodiment, the invention includes the use or administrationof antibodies and antigen binding fragments thereof having bindingspecificity to PACAP that bind the same epitope as Ab1.H, and whichcontain a constant light chain sequence comprising the sequence setforth below:

(SEQ ID NO: 70) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC.

In another embodiment, the invention includes the use or administrationof antibodies and antigen binding fragments thereof having bindingspecificity to PACAP and that contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 44; SEQ ID NO: 46; and SEQ ID NO:48, which correspond to the CDRs, or hypervariable regions, of the heavychain sequence of SEQ ID NO: 41, or which contain the variable heavychain sequence of SEQ ID NO: 42, and/or which further contain one, two,or three of the polypeptide sequences of SEQ ID NO: 64; SEQ ID NO: 66;and SEQ ID NO: 68, which correspond to the CDRs, or hypervariableregions, of the light chain sequence of SEQ ID NO: 61, or which containthe variable light chain sequence of SEQ ID NO: 62, or antibodies orantigen binding fragments thereof containing combinations of sequences,which are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identicalthereto. In another embodiment of the invention, the anti-PACAPantibodies or antigen binding fragments thereof comprise, oralternatively consist of, combinations of one or more of the exemplifiedvariable heavy chain and variable light chain sequences, or the heavychain and light chain sequences set forth above, or sequences that areat least 90% or 95% identical thereto.

The invention further contemplates the use or administration ofanti-PACAP antibodies and antigen binding fragments thereof comprisingone, two, three, or four of the polypeptide sequences of SEQ ID NO: 43;SEQ ID NO: 45; SEQ ID NO: 47; and SEQ ID NO: 49, which correspond to theFRs, or constant regions, of the heavy chain sequence of SEQ ID NO: 41,or the variable heavy chain sequence of SEQ ID NO: 42, and/or one, two,three, or four of the polypeptide sequences of SEQ ID NO: 63; SEQ ID NO:65; SEQ ID NO: 67; and SEQ ID NO: 69, which correspond to the FRs of thelight chain sequence of SEQ ID NO: 61, or the variable light chainsequence of SEQ ID NO: 62, or combinations of these polypeptidesequences, or sequences which are at least 80%, 90%, 95%, 96%, 97%, 98%or 99% identical therewith.

In another embodiment of the invention, the antibodies and antigenbinding fragments thereof useful in the disclosed methods comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem, or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-PACAP antibodies andantigen binding fragments useful in the disclosed methods comprise, oralternatively consist of, the polypeptide sequence of SEQ ID NO: 41, orSEQ ID NO: 42, or polypeptides that are at least 90% or 95% identicalthereto. In another embodiment of the invention, the antibodies andantigen binding fragments thereof useful in the disclosed methodscomprise, or alternatively consist of, the polypeptide sequence of SEQID NO: 61, or SEQ ID NO: 62, or polypeptides that are at least 90% or95% identical thereto.

In a further embodiment of the invention, the antibodies and antigenbinding fragments thereof useful in the disclosed methods have bindingspecificity to PACAP and comprise, or alternatively consist of, one,two, or three of the polypeptide sequences of SEQ ID NO: 44; SEQ ID NO:46; and SEQ ID NO: 48, which correspond to the CDRs, or hypervariableregions, of the heavy chain sequence of SEQ ID NO: 41, or the variableheavy chain sequence of SEQ ID NO: 42, or sequences that are at least90%, or 95% identical thereto.

In a further embodiment of the invention, the antibodies and antigenbinding fragments useful in the disclosed methods have bindingspecificity to PACAP and comprise, or alternatively consist of, one,two, or three of the polypeptide sequences of SEQ ID NO: 64; SEQ ID NO:66; and SEQ ID NO: 68 which correspond to the CDRs, or hypervariableregions, of the light chain sequence of SEQ ID NO: 61, or the variablelight chain sequence of SEQ ID NO: 62, or sequences that are at least90%, or 95% identical thereto.

In a further embodiment of the invention, the antibodies and antigenbinding fragments thereof useful in the disclosed methods have bindingspecificity to PACAP and comprise, or alternatively consist of, one,two, three, or four of the polypeptide sequences of SEQ ID NO: 43; SEQID NO: 45; SEQ ID NO: 47; and SEQ ID NO: 49, which correspond to theFRs, or constant regions, of the heavy chain sequence of SEQ ID NO: 41,or the variable heavy chain sequence of SEQ ID NO: 42, or sequences thatare at least 90% or 95% identical thereto.

In a further embodiment of the invention, the antibodies and antigenbinding fragments thereof useful in the disclosed methods have bindingspecificity to PACAP and comprise, or alternatively consist of, one,two, three, or four of the polypeptide sequences of SEQ ID NO: 63; SEQID NO: 65; SEQ ID NO: 67; and SEQ ID NO: 69, which correspond to the FRsor constant regions, of the light chain sequence of SEQ ID NO: 61, orthe variable light chain sequence of SEQ ID NO: 62, or sequences thatare at least 90% or 95% identical thereto.

The invention also contemplates the use or administration of anti-PACAPantibodies and antigen binding fragments thereof that include one ormore of the antigen binding fragments described herein. In oneembodiment of the invention, antibodies and antigen binding fragmentsthereof useful in the disclosed methods have binding specificity toPACAP and comprise, or alternatively consist of, one, two, three ormore, including all of the following antigen binding fragments: thevariable heavy chain region of SEQ ID NO: 42; the variable light chainregion of SEQ ID NO: 62; the CDRs (SEQ ID NO: 44; SEQ ID NO: 46; and SEQID NO: 48) of the variable heavy chain region of SEQ ID NO: 42; and theCDRs (SEQ ID NO: 64; SEQ ID NO: 66; and SEQ ID NO: 68) of the variablelight chain region of SEQ ID NO: 62, or sequences that are at least 90%or 95% identical thereto.

The invention also contemplates the use or administration of anti-PACAPantibodies and antigen binding fragments thereof that include one ormore of the antigen binding fragments described herein. In oneembodiment of the invention, antigen binding fragments of the antibodiesuseful in the disclosed methods have binding specificity to PACAP andcomprise, or alternatively consist of, one, two, three or more,including all of the following antigen binding fragments: the variableheavy chain region of SEQ ID NO: 42; the variable light chain region ofSEQ ID NO: 62; the FRs (SEQ ID NO: 43; SEQ ID NO: 45; SEQ ID NO: 47; andSEQ ID NO: 49) of the variable heavy chain region of SEQ ID NO: 42; andthe FRs (SEQ ID NO: 63; SEQ ID NO: 65; SEQ ID NO: 67; and SEQ ID NO: 69)of the variable light chain region of SEQ ID NO: 62.

In another embodiment of the invention, the anti-PACAP antibody usefulin the disclosed methods is Ab1.H, comprising, or alternativelyconsisting of, SEQ ID NO: 41 and SEQ ID NO: 61, or SEQ ID NO: 42 and SEQID NO: 62, or an antibody or antigen binding fragment comprising theCDRs of Ab1.H and having at least one of the biological activities setforth herein, or is an anti-PACAP antibody that competes with Ab1.H inbinding PACAP, for instance an antibody containing sequences that are atleast 90%, 95%, 96%, 97%, 98%, or 99% identical to that of Ab1.H, or anantibody that binds to the same or overlapping epitope(s) on PACAP asAb1.H.

In a further embodiment of the invention, anti-PACAP antigen bindingfragments useful in the disclosed methods comprise, or alternativelyconsist of, Fab (fragment antigen binding) fragments having bindingspecificity for PACAP. With respect to antibody Ab1.H, the Fab fragmentpreferably includes the variable heavy chain sequence of SEQ ID NO: 42and the variable light chain sequence of SEQ ID NO: 62, or sequencesthat are at least 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.This embodiment of the invention further includes the use of Fabscontaining additions, deletions, and variants of SEQ ID NO: 42 and/orSEQ ID NO: 62, which retain the binding specificity for PACAP.

In one embodiment of the invention described herein, anti-PACAP Fabfragments useful in the disclosed methods can be produced by enzymaticdigestion (e.g., papain) of Ab1.H. In another embodiment of theinvention, anti-PACAP antibodies such as Ab1.H or Fab fragments thereofuseful in the disclosed methods can be produced via expression inmammalian cells such as CHO, NS0, or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed to theuse or administration of polynucleotides encoding antibody polypeptideshaving binding specificity to PACAP, or PACAP receptors, including theheavy and/or light chains of Ab1.H as well as fragments, variants,combinations of one or more of the FRs, CDRs, the variable heavy chainand variable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them, or sequences that areat least 90% or 95% identical thereto.

In another embodiment, the invention contemplates an isolated anti-PACAPantibody comprising a V_(H) polypeptide sequence of SEQ ID NO: 42, or avariant thereof; and further comprising a V_(L) polypeptide sequence ofSEQ ID NO: 62, or a variant thereof, wherein one or more of theframework region residues (“FR residues”) and/or CDR residues in saidV_(H) or V_(L) polypeptide has been substituted with another amino acidresidue resulting in an anti-PACAP antibody that specifically bindsPACAP. The invention also includes humanized and chimeric forms of theseantibodies. The chimeric and humanized antibodies may include an Fcderived from IgG1, IgG2, IgG3, or IgG4 constant regions.

In one embodiment of the invention, the chimeric or humanized antibodiesor fragments or V_(H) or V_(L) polypeptides originate or are derivedfrom one or more rabbit antibodies, e.g., a rabbit antibody isolatedfrom a clonal rabbit B cell population.

In some aspects, the invention provides a vector comprising a nucleicacid molecule encoding an anti-PACAP antibody or fragment thereof asdisclosed herein. In some embodiments, the invention provides a hostcell comprising a nucleic acid molecule encoding an anti-PACAP antibodyor fragment thereof as disclosed herein.

In some aspects, the invention provides an isolated antibody or antigenbinding fragment thereof that competes for binding to PACAP with anantibody or antigen binding fragment thereof disclosed herein.

In some aspects, the invention provides a nucleic acid molecule encodingan antibody or antigen binding fragment thereof as disclosed herein.

In some aspects, the invention provides a pharmaceutical or diagnosticcomposition comprising at least one antibody or antigen binding fragmentthereof as disclosed herein.

In some aspects, the invention provides a method for treating orpreventing a condition associated with elevated PACAP levels in asubject, comprising administering to a subject in need thereof aneffective amount of at least one isolated antibody or antigen bindingfragment thereof as disclosed herein.

In some aspects, the invention provides a method of inhibiting bindingof PACAP to PAC1-R, VPAC1-R, and/or VPAC2-R in a subject comprisingadministering an effective amount of at least one antibody or antigenbinding fragment thereof as disclosed herein.

In some aspects, the invention provides an antibody or antigen bindingfragment thereof that selectively binds to PACAP, wherein the antibodyor antigen binding fragment thereof binds to PACAP with a K_(D) of lessthan or equal to 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M,5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, or 10⁻¹³ M; preferably, with a K_(D)of less than or equal to 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹²M, or 10⁻¹² M; more preferably, with a K_(D) that is less than about 100pM, less than about 50 pM, less than about 40 pM, less than about 25 pM,less than about 1 pM, between about 10 pM and about 100 pM, betweenabout 1 pM and about 100 pM, or between about 1 pM and about 10 pM.Preferably, the anti-PACAP antibody or antigen binding fragment thereofhas no cross-reactivity or minimal cross-reactivity with VIP.

The inventive antibodies and antigen binding fragments thereof may bemodified post-translationally to add effector moieties such as chemicallinkers, detectable moieties such as for example fluorescent dyes,enzymes, substrates, bioluminescent materials, radioactive materials,and chemiluminescent moieties, or functional moieties such as forexample streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent,and radioactive materials.

Antibodies and antigen binding fragments thereof may also be chemicallymodified to provide additional advantages such as increased solubility,stability and circulating time (in vivo half-life) of the polypeptide,or decreased immunogenicity (See U.S. Pat. No. 4,179,337). The chemicalmoieties for derivatization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, and thelike. The antibodies and fragments thereof may be modified at randompositions within the molecule, or at predetermined positions within themolecule and may include one, two, three, or more attached chemicalmoieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa. Branchedpolyethylene glycols are described, for example, in U.S. Pat. No.5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol., 56:59-72 (1996);Vorobjev et al., Nucleosides and Nucleotides, 18:2745-2750 (1999); andCaliceti et al., Bioconjug. Chem., 10:638-646 (1999), the disclosures ofeach of which are incorporated herein by reference.

There are a number of attachment methods available to those skilled inthe art (See e.g., EP 0 401 384, herein incorporated by reference,disclosing a method of coupling PEG to G-CSF; and Malik et al., Exp.Hematol., 20:1028-1035 (1992) (reporting pegylation of GM-CSF usingtresyl chloride)). For example, polyethylene glycol may be covalentlybound through amino acid residues via a reactive group, such as, a freeamino or carboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule may be bound. The amino acid residueshaving a free amino group may include lysine residues and the N-terminalamino acid residues; those having a free carboxyl group may includeaspartic acid residues glutamic acid residues and the C-terminal aminoacid residue. Sulfhydryl groups may also be used as a reactive group forattaching the polyethylene glycol molecules. Preferred for therapeuticpurposes is attachment at an amino group, such as attachment at theN-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to polypeptides via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) or to more than one type ofamino acid residue (e.g., lysine, histidine, aspartic acid, glutamicacid, cysteine and combinations thereof).

Alternatively, antibodies or antigen binding fragments thereof may haveincreased in vivo half-lives via fusion with albumin (including but notlimited to recombinant human serum albumin or fragments or variantsthereof (See, e.g., U.S. Pat. No. 5,876,969, EP 0 413 622, and U.S. Pat.No. 5,766,883, herein incorporated by reference in their entirety)), orother circulating blood proteins such as transferrin or ferritin. In apreferred embodiment, polypeptides and/or antibodies of the presentinvention (including fragments or variants thereof) are fused with themature form of human serum albumin (i.e., amino acids 1-585 of humanserum albumin as shown in FIGS. 1 and 2 of EP 0 322 094) which is hereinincorporated by reference in its entirety. Polynucleotides encodingfusion proteins of the invention are also encompassed by the invention.

Regarding detectable moieties, further exemplary enzymes include, butare not limited to, horseradish peroxidase, acetylcholinesterase,alkaline phosphatase, beta-galactosidase, and luciferase. Furtherexemplary fluorescent materials include, but are not limited to,rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone,dichlorotriazinylamine, phycoerythrin, and dansyl chloride. Furtherexemplary chemiluminescent moieties include, but are not limited to,luminol. Further exemplary bioluminescent materials include, but are notlimited to, luciferin and aequorin. Further exemplary radioactivematerials include, but are not limited to, Iodine 125 (¹²⁵I) Carbon 14(¹⁴C), Sulfur 35 (³⁵S), Tritium (³H) and Phosphorus 32 (³²P).

Methods are known in the art for conjugating an antibody or antigenbinding fragment thereof to a detectable moiety and the like, such asfor example those methods described by Hunter et al., Nature, 144:945(1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J.Immunol. Meth., 40:219 (1981); and Nygren, J., Histochem. and Cytochem.,30:407 (1982).

Embodiments described herein further include variants and equivalentsthat are substantially homologous to the antibodies, antibody fragments,diabodies, SMIPs, camelbodies, nanobodies, IgNAR, polypeptides, variableregions, and CDRs set forth herein. These may contain, e.g.,conservative substitution mutations, (i.e., the substitution of one ormore amino acids by similar amino acids). For example, conservativesubstitution refers to the substitution of an amino acid with anotherwithin the same general class, e.g., one acidic amino acid with anotheracidic amino acid, one basic amino acid with another basic amino acid,or one neutral amino acid by another neutral amino acid. What isintended by a conservative amino acid substitution is well known in theart.

In another embodiment, the invention contemplates polypeptide sequenceshaving at least 90% or greater sequence homology to any one or more ofthe polypeptide sequences of antigen binding fragments, variable regionsand CDRs set forth herein. More preferably, the invention contemplatespolypeptide sequences having at least 95% or greater sequence homology,even more preferably at least 98% or greater sequence homology, andstill more preferably at least 99% or greater sequence homology to anyone or more of the polypeptide sequences of antigen binding fragments,variable regions, and CDRs set forth herein.

Methods for determining homology between nucleic acid and amino acidsequences are well known to those of ordinary skill in the art.

In another embodiment, the invention further contemplates theabove-recited polypeptide homologs of the antigen binding fragments,variable regions and CDRs set forth herein further having anti-PACAPactivity. Non-limiting examples of anti-PACAP activity are set forthherein, e.g., ability to inhibit PACAP binding to PAC1-R, VPAC1-R,and/or VPAC2-R, thereby resulting in the reduced production of cAMP.

In another embodiment, the invention further contemplates the generationand use of antibodies that bind any of the foregoing sequences,including, but not limited to, anti-idiotypic antibodies. In anexemplary embodiment, such an anti-idiotypic antibody could beadministered to a subject who has received an anti-PACAP antibody tomodulate, reduce, or neutralize, the effect of the anti-PACAP antibody.Such antibodies could also be useful for treatment of an autoimmunedisease characterized by the presence of anti-PACAP antibodies. Afurther exemplary use of such antibodies, e.g., anti-idiotypicantibodies, is for detection of the anti-PACAP antibodies of the presentinvention, for example to monitor the levels of the anti-PACAPantibodies present in a subject's blood or other bodily fluids. Forexample, in one embodiment, the invention provides a method of using theanti-idiotypic antibody to monitor the in vivo levels of said anti-PACAPantibody or antigen binding fragment thereof in a subject or toneutralize said anti-PACAP antibody in a subject being administered saidanti-PACAP antibody or antigen binding fragment thereof.

The present invention also contemplates anti-PACAP antibodies comprisingany of the polypeptide or polynucleotide sequences described hereinsubstituted for any of the other polynucleotide sequences describedherein. For example, without limitation thereto, the present inventioncontemplates antibodies comprising the combination of any of thevariable light chain and variable heavy chain sequences describedherein, and further contemplates antibodies resulting from substitutionof any of the CDR sequences described herein for any of the other CDRsequences described herein.

Exemplary Polynucleotides Encoding Anti-PACAP Antibody Polypeptides

The invention is further directed to polynucleotides encoding antibodypolypeptides having binding specificity to PACAP.

Antibody Ab1.H

In one embodiment, the invention is further directed to the use ofpolynucleotides encoding antibody polypeptides having bindingspecificity to PACAP. In one embodiment of the invention,polynucleotides comprise, or alternatively consist of, thepolynucleotide sequence of SEQ ID NO: 51 which encodes the heavy chainsequence of SEQ ID NO: 41 and which consists of the heavy chain variableregion coding sequence of SEQ ID NO: 52 and the heavy chain constantregion coding sequence of SEQ ID NO: 60.

In another embodiment of the invention, the polynucleotides comprise, oralternatively consist of, the following polynucleotide sequence encodingthe variable heavy chain polypeptide sequence of SEQ ID NO: 42:

(SEQ ID NO: 52) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtagcgctgtaatgaattgggtccgtcaggctccagggaaggggctggagtggatcggaagtattgttgctagtggtaccacatactacgctagctctgctaacggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgctagagggggaggggaatttttcatctggggccaagggaccctcgtcacc gtctcgagc.

In another embodiment of the invention, polynucleotides comprise, oralternatively consist of, the following polynucleotide sequence encodingthe constant heavy chain polypeptide sequence of SEQ ID NO: 50:

(SEQ ID NO: 60) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides comprise, oralternatively consist of, the polynucleotide sequence of SEQ ID NO: 71which encodes the light chain polypeptide sequence of SEQ ID NO: 61 andwhich consists of the light chain variable region coding sequence of SEQID NO: 72 and the light chain constant region coding sequence of SEQ IDNO: 80.

In another embodiment of the invention, polynucleotides comprise, oralternatively consist of, the following polynucleotide sequence encodingthe variable light chain polypeptide sequence of SEQ ID NO: 62:

(SEQ ID NO: 72) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcagtccagtgagagtgtttatagtaactacttatcctggtatcagcagaaaccaggaaaagcccctaagttcctgatctatcaggcatccaatttggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtgcaggcggttatagtgaaaacattgttggtttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides comprise, oralternatively consist of, the following polynucleotide sequence encodingthe constant light chain polypeptide sequence of SEQ ID NO: 70:

(SEQ ID NO: 80) acggtagcggccccatctgtettcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantigen binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one or more of the polynucleotide sequencesof SEQ ID NO: 54; SEQ ID NO: 56; and SEQ ID NO: 58, which correspond topolynucleotides encoding the CDRs, or hypervariable regions, of theheavy chain sequence of SEQ ID NO: 41, or the variable heavy chainsequence of SEQ ID NO: 42, and/or one or more of the polynucleotidesequences of SEQ ID NO: 74; SEQ ID NO: 76 and SEQ ID NO: 78, whichcorrespond to the CDRs, or hypervariable regions, of the light chainsequence of SEQ ID NO: 61, or the variable light chain sequence of SEQID NO: 62, or combinations of these polynucleotide sequences. In anotherembodiment of the invention, the polynucleotides encoding the antibodiesand antigen binding fragments thereof comprise, or alternatively consistof, combinations of polynucleotides encoding one or more of the CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem.

In a further embodiment of the invention, polynucleotides encodingantigen binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one or more of the polynucleotide sequencesof SEQ ID NO: 53; SEQ ID NO: 55; SEQ ID NO: 57; and SEQ ID NO: 59, whichcorrespond to polynucleotides encoding the FRs, or constant regions, ofthe heavy chain sequence of SEQ ID NO: 41, or the variable heavy chainsequence of SEQ ID NO: 42, and/or one or more of the polynucleotidesequences of SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO: 77; and SEQ ID NO:79, which correspond to the FRs, or constant regions, of the light chainsequence of SEQ ID NO: 61, or the variable light chain sequence of SEQID NO: 62, or combinations of these polynucleotide sequences. In anotherembodiment of the invention, the polynucleotides encoding the antibodiesand antigen binding fragments thereof comprise, or alternatively consistof, combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates the use of polynucleotide sequencesincluding one or more of the polynucleotide sequences encoding antigenbinding fragments described herein. In one embodiment of the invention,polynucleotides encoding antigen binding fragments having bindingspecificity to PACAP comprise, or alternatively consist of, one, two,three or more, including all of the following polynucleotides encodingantigen binding fragments: the polynucleotide SEQ ID NO: 51 encoding theheavy chain sequence of SEQ ID NO: 41; the polynucleotide SEQ ID NO: 52encoding the variable heavy chain sequence of SEQ ID NO: 42; thepolynucleotide SEQ ID NO: 71 encoding the light chain sequence of SEQ IDNO: 61; the polynucleotide SEQ ID NO: 72 encoding the variable lightchain sequence of SEQ ID NO: 62; polynucleotides encoding the CDRs (SEQID NO: 54; SEQ ID NO: 56; and SEQ ID NO: 58) of the heavy chain sequenceof SEQ ID NO: 41 or the variable heavy chain sequence of SEQ ID NO: 42;polynucleotides encoding the CDRs (SEQ ID NO: 74; SEQ ID NO: 76; and SEQID NO: 78) of the light chain sequence of SEQ ID NO: 61, or the variablelight chain sequence of SEQ ID NO: 62; polynucleotides encoding the FRs(SEQ ID NO: 53; SEQ ID NO: 55; SEQ ID NO: 57; and SEQ ID NO: 59) of theheavy chain sequence of SEQ ID NO: 41, or the variable heavy chainsequence of SEQ ID NO: 42; and polynucleotides encoding the FRs (SEQ IDNO: 73; SEQ ID NO: 75; SEQ ID NO: 77; and SEQ ID NO: 79) of the lightchain sequence of SEQ ID NO: 61, or the variable light chain sequence ofSEQ ID NO: 62.

In a preferred embodiment of the invention, polynucleotides comprise, oralternatively consist of, polynucleotides encoding Fab fragments havingbinding specificity for PACAP. With respect to antibody Ab1.H, thepolynucleotides encoding the full length Ab1.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 51 encoding theheavy chain sequence of SEQ ID NO: 41, and the polynucleotide SEQ ID NO:71 encoding the light chain sequence of SEQ ID NO: 61.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NS0, HEK-293 cells, or in fungal, insect, or microbialsystems such as yeast cells such as the yeast Pichia. Suitable Pichiaspecies include, but are not limited to, Pichia pastoris. In oneembodiment of the invention described herein, Fab fragments can beproduced by enzymatic digestion (e.g., papain) of Ab1.H followingexpression of the full-length polynucleotides in a suitable host. Inanother embodiment of the invention, anti-PACAP antibodies such as Ab1.Hor Fab fragments thereof can be produced via expression of Ab1.Hpolynucleotides in mammalian cells such as CHO, NS0, or HEK 293 cells,fungal, insect, or microbial systems such as yeast cells (for examplediploid yeast such as diploid Pichia) and other yeast strains. SuitablePichia species include, but are not limited to, Pichia pastoris.

Host cells and vectors comprising said polynucleotides are alsocontemplated.

The invention further contemplates vectors comprising the polynucleotidesequences encoding the variable heavy and light chain polypeptidesequences, as well as the individual CDRs (hypervariable regions), asset forth herein, as well as host cells comprising said vectorsequences. In embodiments of the invention, the host cells are mammaliancells, such as CHO cells. In embodiments of the invention, the hostcells are yeast cells, such as yeast cells of the genus Pichia.

B-Cell Screening and Isolation

In one embodiment, the present invention contemplates the preparationand isolation of a clonal population of antigen-specific B-cells thatmay be used for isolating at least one PACAP antigen-specific cell,which can be used to produce a monoclonal antibody against PACAP, whichis specific to a desired PACAP antigen, or a nucleic acid sequencecorresponding to such an antibody. Methods of preparing and isolatingsaid clonal population of antigen-specific B-cells are taught, forexample, in U.S. Patent Publication No. US2007/0269868 toCarvalho-Jensen et al., the disclosure of which is herein incorporatedby reference in its entirety. Methods of preparing and isolating saidclonal population of antigen-specific B-cells are also taught herein inthe examples. Methods of “enriching” a cell population by size ordensity are known in the art. See, e.g., U.S. Pat. No. 5,627,052. Thesesteps can be used in addition to enriching the cell population byantigen-specificity.

Methods of Humanizing Antibodies

In another embodiment, the present invention contemplates methods forhumanizing antibody heavy and light chains. Methods for humanizingantibody heavy and light chains that may be applied to anti-PACAPantibodies are taught, for example, in U.S. Patent Publication No.US2009/0022659 to Olson et al., and in U.S. Pat. No. 7,935,340 toGarcia-Martinez et al., the disclosures of each of which are hereinincorporated by reference in their entireties.

Methods of Producing Antibodies and Fragments Thereof

In another embodiment, the present invention contemplates methods forproducing anti-PACAP antibodies and fragments thereof. Methods forproducing anti-PACAP antibodies and fragments thereof secreted frompolyploidal, preferably diploid or tetraploid strains of matingcompetent yeast are taught, for example, in U.S. Patent Publication No.US2009/0022659 to Olson et al., and in U.S. Pat. No. 7,935,340 toGarcia-Martinez et al., the disclosures of each of which are hereinincorporated by reference in their entireties.

Other methods of producing antibodies are well known to those ofordinary skill in the art. For example, methods of producing chimericantibodies are now well known in the art (See, for example, U.S. Pat.No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci.U.S.A., 81:8651-55 (1984); Neuberger et al., Nature, 314:268-270 (1985);Boulianne, G. L. et al., Nature, 312:643-46 (1984), the disclosures ofeach of which are herein incorporated by reference in their entireties).

Likewise, other methods of producing humanized antibodies are now wellknown in the art (See, for example, U.S. Pat. Nos. 5,530,101, 5,585,089,5,693,762, and 6,180,370 to Queen et al; U.S. Pat. Nos. 5,225,539 and6,548,640 to Winter; U.S. Pat. Nos. 6,054,297, 6,407,213 and 6,639,055to Carter et al; U.S. Pat. No. 6,632,927 to Adair; Jones, P. T. et al.,Nature, 321:522-525 (1986); Reichmann, L. et al., Nature, 332:323-327(1988); Verhoeyen, M. et al., Science, 239:1534-36 (1988), thedisclosures of each of which are herein incorporated by reference intheir entireties).

Antibody polypeptides of the invention having PACAP binding specificitymay also be produced by constructing, using conventional techniques wellknown to those of ordinary skill in the art, an expression vectorcontaining a promoter (optionally as a component of a eukaryotic orprokaryotic operon) and a DNA sequence encoding an antibody heavy chainin which the DNA sequence encoding the CDRs required for antibodyspecificity is derived from a non-human cell source, preferably a rabbitB-cell source, while the DNA sequence encoding the remaining parts ofthe antibody chain is derived from a human cell source.

A second expression vector is produced using the same conventional meanswell known to those of ordinary skill in the art, said expression vectorcontaining a promoter (optionally as a component of a eukaryotic orprokaryotic operon) and a DNA sequence encoding an antibody light chainin which the DNA sequence encoding the CDRs required for antibodyspecificity is derived from a non-human cell source, preferably a rabbitB-cell source, while the DNA sequence encoding the remaining parts ofthe antibody chain is derived from a human cell source.

The expression vectors are transfected into a host cell by conventiontechniques well known to those of ordinary skill in the art to produce atransfected host cell, said transfected host cell cultured byconventional techniques well known to those of ordinary skill in the artto produce said antibody polypeptides.

The host cell may be co-transfected with the two expression vectorsdescribed above, the first expression vector containing DNA encoding apromoter (optionally as a component of a eukaryotic or prokaryoticoperon) and a light chain-derived polypeptide and the second vectorcontaining DNA encoding a promoter (optionally as a component of aeukaryotic or prokaryotic operon) and a heavy chain-derived polypeptide.The two vectors contain different selectable markers, but preferablyachieve substantially equal expression of the heavy and light chainpolypeptides. Alternatively, a single vector may be used, the vectorincluding DNA encoding both the heavy and light chain polypeptides. Thecoding sequences for the heavy and light chains may comprise cDNA,genomic DNA, or both.

The host cells used to express the antibody polypeptides may be either abacterial cell such as E. coli, or a eukaryotic cell such as P.pastoris. In one embodiment of the invention, a mammalian cell of awell-defined type for this purpose, such as a myeloma cell, a CHO cellline, a NS0 cell line, or a HEK293 cell line may be used.

The general methods by which the vectors may be constructed,transfection methods required to produce the host cell and culturingmethods required to produce the antibody polypeptides from said hostcells all include conventional techniques. Although preferably the cellline used to produce the antibody is a mammalian cell line, any othersuitable cell line, such as a bacterial cell line such as an E.coli-derived bacterial strain, or a yeast cell line, may alternativelybe used.

Similarly, once produced the antibody polypeptides may be purifiedaccording to standard procedures in the art, such as for examplecross-flow filtration, ammonium sulphate precipitation, affinity columnchromatography, hydrophobic interaction chromatography (“HIC”), and thelike.

The antibody polypeptides described herein may also be used for thedesign and synthesis of either peptide or non-peptide mimetics thatwould be useful for the same therapeutic applications as the antibodypolypeptides of the invention (See, for example, Saragobi et al.,Science, 253:792-795 (1991), the contents of which are hereinincorporated by reference in its entirety).

Screening Assays

The invention also includes screening assays designed to assist in theidentification and/or selection of antibodies and antigen bindingfragments thereof having binding specificity for PACAP or a PACAPreceptor, such as PAC1-R, useful in the disclosed methods for treating,ameliorating, or preventing diseases and disorders associated withPACAP, such as photophobia or light aversion, in subjects exhibitingsymptoms of a PACAP associated disease or disorder, particularlyconditions associated with PACAP-associated photophobia or lightaversion, which are identified herein. Exemplary screening methods areuseful for identifying or selecting polypeptides having activities thatameliorate, inhibit, reduce, treat, prevent, relieve, or otherwisediminish the effects of photophobia and/or light aversion or conditionsassociated with photophobia.

In some embodiments, the antibody or fragment thereof disclosed hereinis used as a diagnostic tool. The antibody can be used to assay theamount of PACAP present in a sample and/or subject, for instance asubject being treated for PACAP-associated photophobia or a subjecthaving already been treated for PACAP-associated photophobia. In someembodiments, the diagnostic antibody is not a neutralizing antibody. Insome embodiments, the diagnostic antibody binds to a different epitopethan the neutralizing antibody binds to. In some embodiments, the twoantibodies do not compete with one another.

In some embodiments, the anti-PACAP, or anti-PACAP receptor, antibodiesor antigen binding fragments thereof are used or provided in an assaykit and/or for use in a method for the detection of PACAP in mammaliantissues, or cells in order to screen/diagnose for a disease or disorderassociated with changes in levels of PACAP, or changes in light aversionor PACAP-associated photophobia. The kit comprises an antibody thatbinds PACAP and means for indicating the binding of the antibody withPACAP, if present, and optionally PACAP protein levels. Various meansfor indicating the presence of an antibody can be used. For example,fluorophores, other molecular probes, or enzymes can be linked to theantibody and the presence of the antibody can be observed in a varietyof ways. The method for screening for such disorders can involve the useof the kit, or simply the use of one of the disclosed antibodies, andthe determination of whether the antibody binds to PACAP in a sample. Aswill be appreciated by one of skill in the art, high or elevated levelsof PACAP will result in larger amounts of the antibody binding to PACAPin the sample. Thus, degree of antibody binding can be used to determinehow much PACAP is in a sample. Subjects or samples with an amount ofPACAP that is greater than a predetermined amount (e.g., an amount orrange that a person without a PACAP-related disorder would have, such asa “normal” amount, or an amount typically present in a cohort ofindividuals who exhibit no PACAP-associated photophobia or lightaversion) can be characterized as having a PACAP-mediated disorder,e.g., migraine, headache, pain, photophobia, light aversion, or othercondition.

The present invention further provides for a kit for detecting bindingof an anti-PACAP, or anti-PACAP receptor, antibody of the invention toPACAP, or PACAP receptor, respectively. In particular, the kit may beused to detect the presence of PACAP specifically reactive with ananti-PACAP antibody of the invention, or an immunoreactive fragmentthereof. The kit may also include an antibody bound to a substrate, asecondary antibody reactive with the antigen, and a reagent fordetecting a reaction of the secondary antibody with the antigen. Such akit may be an ELISA kit and may comprise the substrate, primary, andsecondary antibodies when appropriate, and any other necessary reagents,such as detectable moieties, enzyme substrates, and color reagents, forexample, as described herein. The diagnostic kit may also be in the formof an immunoblot kit. The diagnostic kit may also be in the form of achemiluminescent kit (see, for example, Meso Scale Discovery,Gaithersburg, Md.). The diagnostic kit may also be a lanthanide-baseddetection kit (PerkinElmer, San Jose, Calif.).

A skilled clinician would understand that a biological sample includes,but is not limited to, sera, plasma, urine, saliva, mucous, pleuralfluid, synovial fluid and spinal fluid.

Methods of Ameliorating or Reducing Symptoms of, or Treating, orPreventing, Diseases and Disorders Associated with PACAP

In another embodiment of the invention, anti-PACAP antibodies describedherein, or antigen binding fragments thereof, are useful forameliorating or reducing the symptoms of, or treating, or preventing,diseases and disorders associated with PACAP. Non-limiting examples ofsuch symptoms or diseases may be photophobia, light aversion oravoidance, and sensitivity to light. Anti-PACAP antibodies describedherein, or antigen binding fragments thereof, as well as combinations,can also be administered in a therapeutically effective amount topatients in need of treatment of diseases and disorders associated withPACAP in the form of a pharmaceutical composition as described ingreater detail below.

In another embodiment of the invention, anti-PACAP antibodies describedherein, or antigen binding fragments thereof, are useful (either aloneor in combination with another agent) for ameliorating or reducing thesymptoms of, or treating, or preventing a disease or conditionassociated with PACAP, such as photophobia, light aversion or avoidanceand sensitivity to light.

In another embodiment of the invention, anti-PACAP antibodies describedherein, or antigen binding fragments thereof, with or without a secondagent, are useful for ameliorating or reducing the symptoms of, ortreating, or preventing, the following non-limiting listing of diseasesand disorders: migraine (with or without aura), hemiplegic migraines,cluster headaches, migrainous neuralgia, chronic headaches, tensionheadaches, general headaches, hot flush, photophobia, chronic paroxysmalhemicrania, secondary headaches due to an underlying structural problemin the head or neck, cranial neuralgia, sinus headaches (e.g., headacheassociated with sinusitis), allergy-induced headaches or migraines,pain, chronic pain, neuroinflammatory or inflammatory pain,post-operative incision pain, post-surgical pain, trauma-related pain,eye pain, tooth pain, complex regional pain syndrome, cancer pain (e.g.,primary or metastatic bone cancer pain), fracture pain, osteoporoticfracture pain, pain resulting from burn, gout joint pain, painassociated with sickle cell crises, pain associated withtemporomandibular disorders, cirrhosis, hepatitis, neurogenic pain,neuropathic pain, nociceptic pain, visceral pain, trigeminal neuralgia,post-herpetic neuralgia, phantom limb pain, fibromyalgia, menstrualpain, ovarialgia, reflex sympathetic dystrophy, osteoarthritis orrheumatoid arthritis pain, lower back pain, diabetic neuropathy,sciatica, dyspepsia, irritable bowel syndrome, inflammatory boweldisease, Crohn's disease, ileitis, ulcerative colitis, renal colic,dysmenorrhea, cystitis, interstitial cystitis, menstrual period, labor,menopause, pancreatitis, schizophrenia, depression, post-traumaticstress disorder, anxiety disorders, diabetes, autoimmune diabetes,endothelial dysfunction, ischemia, Raynaud's syndrome, coronary heartdisease (“CHD”), coronary artery disease (“CAD”), heart failure,peripheral arterial disease (“PAD”), pulmonary hypertension (“PH”),connective tissue disorders, stroke, Sjögren's syndrome, multiplesclerosis, bronchial hyperreactivity, asthma, bronchitis,bronchodilation, emphysema, chronic obstructive pulmonary disease(“COPD”), inflammatory dermatitis, adenocarcinoma in glandular tissue,blastoma in embryonic tissue of organs, carcinoma in epithelial tissue,leukemia in tissues that form blood cells, lymphoma in lymphatic tissue,myeloma in bone marrow, sarcoma in connective or supportive tissue,adrenal cancer, AIDS-related lymphoma, anemia, bladder cancer, bonecancer, brain cancer, breast cancer, carcinoid tumors, cervical cancer,chemotherapy, colon cancer, cytopenia, endometrial cancer, esophagealcancer, gastric cancer, head cancer, neck cancer, hepatobiliary cancer,kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, Hodgkin'sdisease, non-Hodgkin's, nervous system tumors, oral cancer, ovariancancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer,stomach cancer, testicular cancer, thyroid cancer, urethral cancer,cancer of bone marrow, multiple myeloma, tumors that metastasize to thebone, tumors infiltrating the nerve and hollow viscus, tumors nearneural structures. Further preferably the cancer pain comprises visceralpain, preferably visceral pain which arises from pancreatic cancerand/or metastases in the abdomen. Further preferably the cancer paincomprises somatic pain, preferably somatic pain due to one or more ofmetastasis in the bone, postsurgical pain, sarcomas cancer of theconnective tissue, cancer of bone tissue, cancer of blood-forming cellsof the bone marrow, multiple myeloma, leukemia, primary or secondarybone cancer, acne vulgaris, atopic dermatitis, urticaria, keloids,hypertrophic scars and rosacea, allergic dermatitis, psoriasis,pruritus, neurogenic cutaneous redness, erythema, weight loss, anorexia,sarcoidosis, shock, sepsis, opiate withdrawal syndrome, morphinetolerance, epilepsy, lower urinary tract (“LUT”) disorders such asurinary tract infection, abnormal voiding, urinary urgency, nocturia,urinary incontinence, overactive bladder and for preventing oralleviating the pain associated with such LUT conditions. Preferably,the subject anti-PACAP antibodies and antigen binding fragmentsdescribed herein are useful for ameliorating or reducing the symptomsof, treating, or preventing migraine, headache and a pain associateddisease or condition.

In particular, the subject anti-PACAP antibodies and antigen bindingfragments can also be useful for ameliorating or reducing the symptomsof, treating, or preventing photophobia, occurring with a headacheand/or migraine as well as occurring independent of a headache and/or amigraine.

Migraineurs typically develop worsening pain and migraine symptoms whenexposed to light, a phenomenon known as photophobia. Photophobia is alsocommon in ocular disorders, such as iritis and uveitis, and intracranialdisorders, such as meningitis. In the classic visual pathway, lightactivates rods and cones in the retina, which activate retinal ganglioncells that project via the optic nerve, to the lateral geniculatenucleus, superior colliculus, and then the visual cortex. This pathwayincludes image-forming and non-image-forming data. A new pathway(non-image-forming information) allows maintenance of normal circadianrhythms via the suprachiasmatic nucleus and is regulated byintrinsically photosensitive retinal ganglion cells (ipRGCs). TheseipRGCs are independent of the rods and cones and contain melanopsin, aphotopigment.

Noseda, R. et al., Nat. Neurosci., 13:239-245 (2010) studied blindindividuals who had migraine and correlated these findings with ratmodels involving tracing of ipRGC projections to areas in perception ofpain from the dura. Of the blind patients with migraine, 6 had no lightperception due to severe optic nerve damage or bilateral enucleation.These subjects experienced abnormal sleep patterns and poor pupillarylight responses. Their migraines did not worsen with light exposure. Incontrast, 14 blind subjects who were able to detect light despiteminimal perception of images had normal sleep patterns and a normalpupillary light reflex. Despite widespread rod and cone degeneration,these patients had worsening migraine symptoms with light exposureduring migraine attacks, suggesting that ipRGCs, and not rods and cones,are important in photophobia.

These retinal projections of non-image-forming brain areas project tothe contralateral dorsocaudal region of the posterior thalamus, asdemonstrated by anterograde tracing in the rat. ipRGC input to this areamodulates dura-sensitive pain neurons, which also project to thisregion. Thalamic neurons, dually sensitive to dural pain and lightinput, project widely to multiple cortical regions, including theprimary somatosensory cortex, the primary and secondary motor cortices,the parietal association cortex, and the primary and secondary visualcortices. These cortical projections may help explain other commonmigraine symptoms, in addition to photophobia, such as motor weakness orincoordination, visual disturbances, and poor concentration.

Photophobia also accompanies other less frequent but likewise disablingconditions, such as cluster headache and other trigeminal autonomiccephalalgias and blepharospasm. The mechanisms underlying photophobiainvolve the trigeminal system. Photophobia in blind patients suggestscontributions from a nonvisual pathway. In addition, trigeminalautonomic cephalalgias, a less common group of primary headachedisorders, are characterized by unilateral trigeminal-mediated painfrequently associated with ipsilateral photophobia.

Common causes of photophobia include migraine headaches, cataracts, orsevere ophthalmologic diseases such as uveitis or corneal abrasion. Amore extensive list of disorders associated with photophobia includeseye related causes such as achromatopsia, aniridia, anticholinergicdrugs may cause photophobia by paralyzing the iris sphincter muscle,aphakia (absence of the lens of the eye), buphthalmos (abnormally narrowangle between the cornea and iris), cataracts, cone dystrophy,congenital abnormalities of the eye, viral conjunctivitis (“pink eye”),corneal abrasion, corneal dystrophy, corneal ulcer, disruption of thecorneal epithelium, such as that caused by a corneal foreign body orkeratitis, ectopia lentis, endophthalmitis, eye trauma caused bydisease, injury, or infection such as chalazion, episcleritis, glaucoma,keratoconus, or optic nerve hypoplasia, hydrophthalmos, or congenitalglaucoma iritis, optic neuritis, pigment dispersion syndrome, pupillarydilation (naturally or chemically induced), retinal detachment, scarringof the cornea or sclera and uveitis.

In addition, photophobia has nervous-system-related or neurologicalcauses including: autism spectrum disorders, Chiari malformation,dyslexia, encephalitis including myalgic encephalomyelitis aka chronicfatigue syndrome, meningitis, subarachnoid hemorrhage, tumor of theposterior cranial fossa, as well as other causes such as ankylosingspondylitis, albinism, ariboflavinosis, benzodiazepines (long term useof or withdrawal from benzodiazepines), chemotherapy, chikungunya,cystinosis, Ehlers-Danlos syndrome, hangover, influenza, infectiousmononucleosis, magnesium deficiency, mercury poisoning, migraine,rabies, and tyrosinemia type II, also known as “Richner-Hanhartsyndrome”.

Additionally, it is known that photophobia is elevated in depression,bipolar disorder and agoraphobia.

The subject anti-PACAP antibodies and antigen binding fragmentsdescribed herein can be effective for treating or preventing photophobiain any of these conditions, preferably, in a subject with post-traumaticstress disorder (“PTSD”) or in a subject with traumatic brain injury.

Headaches may be classified by cause, as discussed below.

Primary Headaches.

A primary headache is caused by problems with or overactivity ofpain-sensitive structures in the head. A primary headache is generallynot considered to be a symptom of an underlying disease. Instead,chemical activity in the brain, the nerves or blood vessels of the headoutside the skull, or muscles of the head and neck, or some combinationof these factors, may play a role in primary headaches. Some people maycarry genes that make them more likely to develop such headaches.Exemplary common primary headaches include, but are not limited to,cluster headache; tension headache (or tension-type headache); andtrigeminal autonomic cephalalgia (“TAC”), including paroxysmalhemicrania. There are other headache patterns that may be consideredtypes of primary headache, e.g., chronic daily headaches, coughheadaches, exercise headaches, and sex headaches. These headaches areless common and have distinct features, such as an unusual duration orpain associated with a certain activity. Although these headaches aregenerally considered primary, each of them could be a symptom of anunderlying disease. Additionally, some primary headaches can betriggered by lifestyle factors, including: alcohol; certain foods (e.g.,processed meats that contain nitrates); changes in sleep or lack ofsleep; poor posture; skipped meals; and stress.

Secondary Headaches.

A secondary headache is a symptom of a disease that can activate thepain-sensitive nerves of the head. Any number of conditions, which canvary greatly in severity, may cause secondary headaches. Exemplarysources of secondary headaches include, but are not limited to, acutesinusitis; arterial tears (carotid or vertebral dissections); venousthrombosis in the brain; brain aneurysm; brain arteriovenousmalformation; carbon monoxide poisoning; Chiari malformation;concussion; dehydration; dental problems; ear infection (middle ear);encephalitis; giant cell arteritis; glaucoma; hangovers; influenza(flu); intracranial hematoma; medications to treat other disorders;meningitis; monosodium glutamate (“MSG”); overuse of pain medication;panic attacks; post-concussion syndrome; pressure from tight-fittingheadgear, e.g., helmet or goggles; pseudotumor cerebri; toxoplasmosis;and trigeminal neuralgia. Specific types of secondary headaches include,but are not limited to, external compression headaches (a result ofpressure-causing headgear); ice cream headaches (commonly called “brainfreeze”); rebound headaches (caused by overuse of pain medication);sinus headaches (caused by inflammation and congestion in sinuscavities); spinal headaches (caused by low levels of cerebrospinalfluid, possibly the result of trauma, spinal tap or spinal anesthesia);and thunderclap headaches (a group of disorders that involves sudden,severe headaches).

Exemplary, non-limiting pain associated diseases and disorders that canbe treated and/or prevented by the administration of the anti-PACAPantibodies of the present invention include, pain resulting from anycondition associated with neurogenic, neuropathic, inflammatory, ornociceptic pain. Preferably, the pain-associated disorder will beassociated with increased PACAP at the pain site.

In certain embodiments, the pain associated disorder to be treated iscancer pain arising from malignancy or from cancer selected from one ormore of: adenocarcinoma in glandular tissue, blastoma in embryonictissue of organs, carcinoma in epithelial tissue, leukemia in tissuesthat form blood cells, lymphoma in lymphatic tissue, myeloma in bonemarrow, sarcoma in connective or supportive tissue, adrenal cancer,AIDS-related lymphoma, anemia, bladder cancer, bone cancer, braincancer, breast cancer, carcinoid tumors, cervical cancer, chemotherapy,colon cancer, cytopenia, endometrial cancer, esophageal cancer, gastriccancer, head cancer, neck cancer, hepatobiliary cancer, kidney cancer,leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's disease,non-Hodgkin's, nervous system tumors, oral cancer, ovarian cancer,pancreatic cancer, prostate cancer, rectal cancer, skin cancer, stomachcancer, testicular cancer, thyroid cancer, urethral cancer, cancer ofbone marrow, multiple myeloma, tumors that metastasize to the bone,tumors infiltrating the nerve and hollow viscus, tumors near neuralstructures. Further preferably the cancer pain comprises visceral pain,preferably visceral pain which arises from pancreatic cancer and/ormetastases in the abdomen. Further preferably the cancer pain comprisessomatic pain, preferably somatic pain due to one or more of metastasisin the bone, postsurgical pain, sarcomas cancer of the connectivetissue, cancer of bone tissue, cancer of blood-forming cells of the bonemarrow, multiple myeloma, leukemia, primary or secondary bone cancer.

In other embodiments, the pain associated condition to be treated isassociated with neuropathic pain and included, by way of example,trigeminal neuralgia, post-herpetic neuralgia, phantom limb pain,fibromyalgia, and reflex sympathetic dystrophy are preferably treated.

Further exemplary pain associated diseases or conditions, include butare not limited to, general pain, chronic pain, inflammatory pain,post-operative incision pain, post-surgical pain, trauma-related pain,lower back pain, eye pain, tooth pain, complex regional pain syndrome,cancer pain (e.g., primary or metastatic bone cancer pain), fracturepain, osteoporotic fracture pain, pain resulting from burn, gout jointpain, pain associated with sickle cell crises, pain associated withtemporomandibular disorders, cirrhosis, hepatitis, neurogenic pain,neuropathic pain, nociceptic pain, visceral pain, trigeminal neuralgia,post-herpetic neuralgia, phantom limb pain, fibromyalgia, menstrualpain, ovarialgia, reflex sympathetic dystrophy, osteoarthritis orrheumatoid arthritis pain, lower back pain, diabetic neuropathy,sciatica, dyspepsia, irritable bowel syndrome, inflammatory boweldisease, Crohn's disease, ileitis, ulcerative colitis, renal colic,dysmenorrhea, cystitis, interstitial cystitis, menstrual period, labor,menopause, pancreatitis, schizophrenia, depression, post-traumaticstress disorder, anxiety disorders, diabetes, autoimmune diabetes,endothelial dysfunction, ischemia, Raynaud's syndrome, coronary heartdisease (“CHD”), coronary artery disease (“CAD”), heart failure,peripheral arterial disease (“PAD”), pulmonary hypertension (“PH”),connective tissue disorders, stroke, Sjögren's syndrome, multiplesclerosis, overactive bladder, bronchial hyperreactivity, asthma,bronchitis, bronchodilation, emphysema, chronic obstructive pulmonarydisease (“COPD”), inflammatory dermatitis, acne vulgaris, atopicdermatitis, urticaria, keloids, hypertrophic scars and rosacea, allergicdermatitis, psoriasis, puritus, neurogenic cutaneous redness, erythema,sarcoidosis, shock, sepsis, and opiate withdrawal syndrome.

Thus, the present invention includes methods of treating, preventing,and/or ameliorating any disease or disorder associated with PACAPactivity or PACAP upregulation (including any of the above mentionedexemplary pain associated diseases, disorders and conditions) throughuse of the antibodies and antigen binding fragments of the invention.

Also, the subject PACAP antibodies and antigen binding fragments may beused alone or in conjunction with other active agents, e.g., opioids andnon-opioid analgesics such as NSAIDs to elicit analgesia or topotentiate the efficacy of another analgesic.

The subject antibodies potentially may be combined with any opioidanalgesic or NSAID or other analgesic, potentially another antibody oranother biologic such as, e.g., an anti-NGF or anti-CGRP or anti-CGRP-Rantibody or antibody fragment or NGF, CGRP or CGRP-R polypeptidefragment or conjugate, in order to increase or enhance pain management.This may allow for such analgesic compounds to be administered forlonger duration or at reduced dosages thereby potentially alleviatingadverse side effects associated therewith.

Of particular interest is the co-administration of the subjectanti-PACAP antibodies and antibody fragments with an anti-CGRP antibody(e.g., ALD403) or an anti-CGRP-R antibody or antibody fragment and,moreover, the use of the subject anti-PACAP antibodies and antibodyfragments to treat subjects that previously received an anti-CGRP oranti-CGRP-R antibody or antibody fragment. For example, the previouslytreated subject (who previously received at least one anti-CGRP oranti-CGRP-R antibody or antibody fragment administration) may be amigraineur who did not adequately respond to anti-CGRP or anti-CGRP-Rantibody treatment (“poor responder”) and/or has elicited an immuneresponse to the anti-CGRP or anti-CGRP-R antibody or antibody fragment.

Likewise, the co-administration of the subject anti-PACAP antibodies andantigen binding fragments with BOTOX® (Botulinum toxin) is also ofparticular interest, e.g., in treating a migraineur. In some instances,the migraineur may not have adequately responded to previous treatments(“poor responder”) and/or has elicited an immune response to theprevious treatment.

In some embodiments, aspirin and/or acetaminophen may be taken inconjunction with the subject anti-PACAP antibody or antigen bindingfragment. Aspirin is another type of non-steroidal anti-inflammatorycompound.

The subject to which the pharmaceutical formulation is administered canbe, e.g., any human or non-human animal that is in need of suchtreatment, prevention and/or amelioration, or who would otherwisebenefit from the inhibition or attenuation of PACAP-mediated activity.For example, the subject can be an individual that is diagnosed with, orwho is deemed to be at risk of being afflicted by any of theaforementioned diseases or disorders. The present invention furtherincludes the use of any of the pharmaceutical formulations disclosedherein in the manufacture of a medicament for the treatment, preventionand/or amelioration of any disease or disorder associated with PACAPactivity (including any of the above mentioned exemplary diseases,disorders and conditions).

Administration

In one embodiment of the invention, the anti-PACAP antibodies describedherein, or PACAP binding fragments thereof, as well as combinations ofsaid antibodies or antigen binding fragments thereof, are administeredto a subject at a concentration of between 0.1 mg/ml and about any oneof 0.5, 1, 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200mg/ml, +/−10% error.

In another embodiment of the invention, the anti-PACAP antibodies andfragments thereof described herein are administered to a subject at adose of between about 0.01 and 100.0 or 200.0 mg/kg of body weight ofthe recipient subject. In certain embodiments, depending on the type andseverity of the PACAP-related disease, about 1 μg/kg to 50 mg/kg (e.g.,0.1-20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. In anotherembodiment, about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) ofantibody is an initial candidate dosage for administration to thepatient. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on several factors, e.g., the particular mammalbeing treated, the clinical condition of the individual patient, thecause of the disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. However, other dosage regimens may beuseful.

For example, in addition to the relative dosages (mg/kg) discussedherein, the subject anti-PACAP antibodies and antigen binding fragmentsthereof can be administered to a subject at an absolute dose (mg).Accordingly, in one embodiment of the invention, the anti-PACAPantibodies and antigen binding fragments thereof described herein areadministered to a subject at a dose of between about 1 microgram andabout 1000 milligrams regardless of the route of administration.

In a preferred embodiment of the invention, the anti-PACAP antibodiesdescribed herein, or anti-PACAP antigen binding fragments thereof, aswell as combinations of said antibodies or antigen binding fragmentsthereof, are administered to a recipient subject with a frequency ofonce every twenty-six weeks or less, such as once every sixteen weeks orless, once every eight weeks or less, once every four weeks or less,once every two weeks or less, once every week or less, or once daily orless.

According to preferred embodiments, the antibody containing medicamentor pharmaceutical composition is peripherally administered to a subjectvia a route selected from one or more of: orally, sublingually,buccally, topically, rectally, via inhalation, transdermally,subcutaneously, intravenously, intra-arterially, or intramuscularly, viaintracardiac administration, intraosseously, intradermally,intraperitoneally, transmucosally, vaginally, intravitreally,epicutaneously, intra-articularly, peri-articularly, or locally.

Fab fragments may be administered every two weeks or less, every week orless, once daily or less, multiple times per day, and/or every fewhours. In one embodiment of the invention, a patient receives Fabfragments of 0.1 mg/kg to 40 mg/kg per day given in divided doses of 1to 6 times a day, or in a continuous perfusion form, effective to obtaindesired results.

It is to be understood that the concentration of the antibody or Fabadministered to a given patient may be greater or lower than theexemplary administration concentrations set forth above.

A person of skill in the art would be able to determine an effectivedosage and frequency of administration through routine experimentation,for example guided by the disclosure herein and the teachings in,Goodman & Gilman's The Pharmacological Basis of Therapeutics, Brunton,L. L. et al. editors, 11^(th) edition, New York, N.Y.: McGraw-Hill(2006); Howland, R. D. et al., Pharmacology, Volume 864, Lippincott'sillustrated reviews., Philadelphia, Pa.: Lippincott Williams & Wilkins(2006); and Golan, D. E., Principles of pharmacology: thepathophysiologic basis of drug therapy, Philadelphia, Pa.: LippincottWilliams & Wilkins (2007).

In another embodiment of the invention, the anti-PACAP antibodiesdescribed herein, or PACAP binding fragments thereof, as well ascombinations of said antibodies or antigen binding fragments thereof,are administered to a subject in a pharmaceutical formulation. In apreferred embodiment, the subject is a human.

A “pharmaceutical composition” or “medicament” refers to a chemical orbiological composition suitable for administration to a subject,preferably a mammal, more preferably a human. Such compositions may bespecifically formulated for administration via one or more of a numberof routes, including but not limited to buccal, epicutaneous, epidural,inhalation, intraarterial, intracardial, intracerebroventricular,intradermal, intramuscular, intranasal, intraocular, intraperitoneal,intraspinal, intrathecal, intravenous, oral, parenteral, rectally via anenema or suppository, subcutaneous, subdermal, sublingual, transdermal,and transmucosal. In addition, administration can occur by means ofinjection, powder, liquid, gel, drops, or other means of administration.

In one embodiment of the invention, the anti-PACAP antibodies describedherein, or PACAP binding fragments thereof, as well as combinations ofsaid antibodies or antigen binding fragments thereof, may be optionallyadministered in combination with one or more active agents. Such activeagents include analgesic, anti-histamine, antipyretic,anti-inflammatory, antibiotic, antiviral, and anti-cytokine agents.Active agents include agonists, antagonists, and modulators of TNF-α,IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-α, IFN-γ, BAFF,CXCL13, IP-10, VEGF, EPO, EGF, HRG, Hepatocyte Growth Factor (“HGF”),Hepcidin, NGF, CGRP including antibodies reactive against any of theforegoing, and antibodies reactive against any of their receptors.Active agents also include but are not limited to 2-arylpropionic acids,aceclofenac, acemetacin, acetylsalicylic acid (aspirin), alclofenac,alminoprofen, amoxiprin, ampyrone, arylalkanoic acids, azapropazone,benorylate/benorilate, benoxaprofen, bromfenac, carprofen, celecoxib,choline magnesium salicylate, clofezone, COX-2 inhibitors, dexibuprofen,dexketoprofen, diclofenac, diflunisal, droxicam, ethenzamide, etodolac,etoricoxib, faislamine, fenamic acids, fenbufen, fenoprofen, flufenamicacid, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indomethacin,indoprofen, kebuzone, ketoprofen, ketorolac, lornoxicam, loxoprofen,lumiracoxib, magnesium salicylate, meclofenamic acid, mefenamic acid,meloxicam, metamizole, methyl salicylate, mofebutazone, nabumetone,naproxen, N-arylanthranilic acids, NGF, oxametacin, oxaprozin, oxicams,oxyphenbutazone, oxytocin, parecoxib, phenazone, phenylbutazone,phenylbutazone, piroxicam, pirprofen, profens, proglumetacin,pyrazolidine derivatives, rofecoxib, salicyl salicylate, salicylamide,salicylates, substance P, sulfinpyrazone, sulindac, suprofen, tenoxicam,tiaprofenic acid, tolfenamic acid, tolmetin, and valdecoxib. Forinstance, the selected anti-PACAP antibodies, or PACAP-binding fragmentsthereof, as well as combinations of these antibodies or antigen bindingfragments, can be optionally administered in combination with oxytocin,for instance administered in a nasal formulation, for intranasaldelivery.

An anti-histamine can be any compound that opposes the action ofhistamine or its release from cells (e.g., mast cells). Anti-histaminesinclude but are not limited to acrivastine, astemizole, azatadine,azelastine, betatastine, brompheniramine, buclizine, cetirizine,cetirizine analogues, chlorpheniramine, clemastine, CS 560,cyproheptadine, desloratadine, dexchlorpheniramine, ebastine,epinastine, fexofenadine, HSR 609, hydroxyzine, levocabastine,loratadine, methscopolamine, mizolastine, norastemizole, phenindamine,promethazine, pyrilamine, terfenadine, and tranilast.

Antibiotics include but are not limited to amikacin, aminoglycosides,amoxicillin, ampicillin, ansamycins, arsphenamine, azithromycin,azlocillin, aztreonam, bacitracin, carbacephem, carbapenems,carbenicillin, cefaclor, cefadroxil, cefalexin, cefalothin, cefalotin,cefamandole, cefazolin, cefdinir, cefditoren, cefepime, cefixime,cefoperazone, cefotaxime, cefoxitin, cefpodoxime, cefprozil,ceftazidime, ceftibuten, ceftizoxime, ceftobiprole, ceftriaxone,cefuroxime, cephalosporins, chloramphenicol, cilastatin, ciprofloxacin,clarithromycin, clindamycin, cloxacillin, colistin, co-trimoxazole,dalfopristin, demeclocycline, dicloxacillin, dirithromycin, doripenem,doxycycline, enoxacin, ertapenem, erythromycin, ethambutol,flucloxacillin, fosfomycin, furazolidone, fusidic acid, gatifloxacin,geldanamycin, gentamicin, glycopeptides, herbimycin, imipenem,isoniazid, kanamycin, levofloxacin, lincomycin, linezolid, lomefloxacin,loracarbef, macrolides, mafenide, meropenem, methicillin, metronidazole,mezlocillin, minocycline, monobactams, moxifloxacin, mupirocin,nafcillin, neomycin, netilmicin, nitrofurantoin, norfloxacin, ofloxacin,oxacillin, oxytetracycline, paromomycin, penicillin, penicillins,piperacillin, platensimycin, polymyxin B, polypeptides, prontosil,pyrazinamide, quinolones, quinupristin, rifampicin, rifampin,roxithromycin, spectinomycin, streptomycin, sulfacetamide,sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole,sulfonamides, teicoplanin, telithromycin, tetracycline, tetracyclines,ticarcillin, tinidazole, tobramycin, trimethoprim,trimethoprim-sulfamethoxazole, troleandomycin, trovafloxacin, andvancomycin.

Active agents also include aldosterone, beclomethasone, betamethasone,corticosteroids, cortisol, cortisone acetate, deoxycorticosteroneacetate, dexamethasone, fludrocortisone acetate, glucocorticoids,hydrocortisone, methylprednisolone, prednisolone, prednisone, steroids,and triamcinolone. Any suitable combination of these active agents isalso contemplated.

A “pharmaceutical excipient” or a “pharmaceutically acceptableexcipient” is a carrier, usually a liquid, in which an activetherapeutic agent is formulated. In one embodiment of the invention, theactive therapeutic agent is a humanized antibody described herein, orone or more fragments thereof. The excipient generally does not provideany pharmacological activity to the formulation, though it may providechemical and/or biological stability, and release characteristics.Exemplary formulations can be found, for example, in Remington'sPharmaceutical Sciences, Gennaro, A. editor, 19^(th) edition,Philadelphia, Pa.: Williams and Wilkins (1995), which is incorporated byreference.

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic, and absorption delaying agents that arephysiologically compatible. In one embodiment, the carrier is suitablefor parenteral administration. Alternatively, the carrier can besuitable for intravenous, intraperitoneal, intramuscular, or sublingualadministration. Pharmaceutically acceptable carriers include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the pharmaceutical compositions of the invention iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The invention contemplatesthat the pharmaceutical composition is present in lyophilized form. Thecomposition can be formulated as a solution, microemulsion, liposome, orother ordered structure suitable to high drug concentration. The carriercan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol), and suitable mixtures thereof. The inventionfurther contemplates the inclusion of a stabilizer in the pharmaceuticalcomposition. The proper fluidity can be maintained, for example, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants.

In many cases, it will be preferable to include isotonic agents, forexample, sugars, polyalcohols such as mannitol and sorbitol, or sodiumchloride in the composition. Absorption of the injectable compositionscan be prolonged by including an agent that delays absorption, forexample, monostearate salts and gelatin. Moreover, the alkalinepolypeptide can be formulated in a time-release formulation, for examplein a composition that includes a slow release polymer. The activecompounds can be prepared with carriers that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid, polylactic and polyglycolic copolymers(“PLG”). Many methods for the preparation of such formulations are knownto those skilled in the art.

For each of the recited embodiments, the compounds can be administeredby a variety of dosage forms. Any biologically acceptable dosage formknown to persons of ordinary skill in the art, and combinations thereof,are contemplated. Examples of such dosage forms include, withoutlimitation, reconstitutable powders, elixirs, liquids, solutions,suspensions, emulsions, powders, granules, particles, microparticles,dispersible granules, cachets, inhalants, aerosol inhalants, patches,particle inhalants, implants, depot implants, injectables (includingsubcutaneous, intramuscular, intravenous, and intradermal), infusions,and combinations thereof.

The above description of various illustrated embodiments of theinvention is not intended to be exhaustive or to limit the invention tothe precise form disclosed. While specific embodiments of, and examplesfor, the invention are described herein for illustrative purposes,various equivalent modifications are possible within the scope of theinvention, as those skilled in the relevant art will recognize. Theteachings provided herein of the invention can be applied to otherpurposes, other than the examples described above.

These and other changes can be made to the invention in light of theabove detailed description. In general, in the following claims, theterms used should not be construed to limit the invention to thespecific embodiments disclosed in the specification and the claims.Accordingly, the invention is not limited by the disclosure, but insteadthe scope of the invention is to be determined entirely by the followingclaims.

The invention may be practiced in ways other than those particularlydescribed in the foregoing description and examples. Numerousmodifications and variations of the invention are possible in light ofthe above teachings and, therefore, are within the scope of the appendedclaims.

Certain teachings related to methods for obtaining a clonal populationof antigen-specific B-cells were disclosed in U.S. Patent PublicationNo. US2013/0316353, the disclosure of which is herein incorporated byreference in its entirety.

Certain teachings related to humanization of rabbit-derived monoclonalantibodies and preferred sequence modifications to maintainantigen-binding affinity were disclosed in International Publication No.WO 2008/144757, entitled Novel Rabbit Antibody Humanization Methods andHumanized Rabbit Antibodies, filed May 21, 2008, the disclosure of whichis herein incorporated by reference in its entirety.

Certain teachings related to producing antibodies or fragments thereofusing mating competent yeast and corresponding methods were disclosed inU.S. Patent Publication No. US2006/0270045, the disclosure of which isherein incorporated by reference in its entirety.

Certain teachings related to producing antibodies or fragments thereofin Pichia and preferred methods for obtaining and purifying antibodiesare also disclosed in U.S. Patent Publication Nos. 2014/0288272;2014/0287952; 2013/0055888; and 2012/0277408, the disclosures of each ofwhich are herein incorporated by reference in their entirety.

Certain teachings related to producing antibodies or fragments thereofin CHO cells and exemplary methods for obtaining and purifyingantibodies are also disclosed in U.S. Pat. No. 7,932,087; 2009/0285795;U.S. Pat. No. 9,090,672; and 2010/0221781; the disclosures of each ofwhich are herein incorporated by reference in their entirety.

Certain anti-PACAP antibody polynucleotides and polypeptides aredisclosed in the sequence listing accompanying this patent applicationfiling, and the disclosure of said sequence listing is hereinincorporated by reference in its entirety.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, manuals, books, or otherdisclosures) in the Background of the Invention, Detailed Description,and Examples is herein incorporated by reference in their entireties.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.), but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXAMPLES Example 1: Preparation of Antibodies that Selectively BindPACAP

By using an antibody selection protocol substantially as describedherein, a panel of antibodies specific to PACAP38 and PACAP27, and apanel of antibodies specific to PACAP38 only, were produced.

Immunization Strategy

Rabbits were immunized with PACAP38 (American Peptide, Vista, Calif.)(SEQ ID NO: 1241). Peptides were prepared for immunization as follows. A0.15 ml volume of 10 mg/ml keyhole limpet hemocyanin (“KLH”) dissolvedin Dulbecco's phosphate buffered saline (“DPBS”) supplemented to 1M NaClwas combined with 1.0 ml of 1 mg/ml peptide (dissolved in deionizedwater). Then 1.0 ml of 40 mM carbodiimide was added prior to a 12-hourincubation at room temperature with gentle mixing. Excess carbodiimideand unconjugated peptide were removed by dialysis to DPBS prior tosterile filtration. Next unconjugated peptide equal to the initial massof KLH was added prior to preparation for injection into rabbits.Alternatively, equal masses of sterile KLH and peptide were mixedwithout carbodiimide chemistry.

Immunizations were performed by diluting 200 μg of antigen to 0.5 mlwith DPBS and mixing with an equal volume of complete Freund's adjuvantfor subcutaneous 1 ml injection at Day 1.

Boost injections of 100 μg were performed with incomplete Freund'sadjuvant at Days 21 and 42.

Antibody Selection Functional Titer Assessment

To identify antibodies that neutralize PACAP38 (SEQ ID NO: 1241) inducedsignaling via PAC1-R, polyclonal antibody solutions were first purifiedvia Protein A and dialyzed into a neutral buffer. Briefly, antibodysolutions were incubated with PACAP38 (SEQ ID NO: 1241) at 4× the finalconcentration (100 pM) for 1 hr. While the antibody/antigen complexeswere incubated, PAC1-R expressing PC-12 cells (Japanese Collection ofResearch Bioresources Cell Bank) were washed and re-suspended at 2×10⁶cells per ml in cell culture media. Cells (10 μl) and antigen/antibodycomplex (40 μl) were transferred to a homogenous time resolvedfluorescence (“HTRF”) plate and shaken at room temperature for 30 min.Following the incubation, 20 μl of (1:20 diluted) Eu³⁺ cryptate-labeledmAb anti-cAMP and 20 μl of (1:20 diluted) d2-labeled cAMP in lysisbuffer were added, and the plate was incubated for 1 hr while shaking.Following incubation, plates were read (excitation 330 nm, emission620/665 nm), and a ratio of 620:665 signal was determined.

Tissue Harvesting

Once acceptable titers were established, the rabbit(s) were sacrificed.Spleen, lymph nodes, and whole blood were harvested and processed asfollows:

Spleen and lymph nodes were processed into a single cell suspension bydisassociating the tissue and pushing through sterile wire mesh at 70 μm(Thermo Fisher Scientific, Waltham, Mass.) with a plunger of a 20 ccsyringe. Cells were collected in phosphate buffered saline (“PBS”).Cells were then washed twice by centrifugation. After the last wash,cell density was determined by trypan blue. Cells were centrifuged at1500 RPM for 10 minutes; the supernatant was then discarded. Cells wereresuspended in the appropriate volume of 10% dimethyl sulfoxide (“DMSO”,Sigma-Aldrich Co., St. Louis, Mo.) in fetal bovine serum (“FBS”HYCLONE™, GE Healthcare Life Sciences, Marlborough, Mass.) and dispensedat 1 ml/vial. Vials were stored at −70° C. in a slow freezing chamberfor 24 hours and stored in liquid nitrogen.

Peripheral blood mononuclear cells (“PBMCs”) were isolated by mixingwhole blood with equal parts of PBS. 35 ml of the whole blood mixturewas carefully layered onto 8 ml of LYMPHOLYTE® Rabbit (CedarlaneLaboratories, Burlington, Ontario) into a 45 ml conical tube (Corning,Corning, N.Y.) and centrifuged for 30 minutes at 2500 RPM at roomtemperature without brakes. After centrifugation, the PBMC layers werecarefully removed using a glass Pasteur pipette (VWR International,Radnor, Pa.), combined, and placed into a clean 50 ml vial. Cells werewashed twice with PBS by centrifugation at 1500 RPM for 10 minutes atroom temperature, and cell density was determined by trypan bluestaining. After the last wash, cells were resuspended in an appropriatevolume of 10% DMSO/FBS medium and frozen as described above.

B-Cell Selection, Enrichment, and Culture Conditions

On the day of setting up B-cell culture, PBMC, splenocyte, or lymph nodevials were thawed for use. Vials were removed from liquid nitrogen tankand placed in a 37° C. water bath until thawed. Contents of vials weretransferred into 15 ml conical centrifuge tube (Corning, Inc., Corning,N.Y.) and 10 ml of modified RPMI was slowly added to the tube. Cellswere centrifuged for 5 minutes at 2000 RPM, and the supernatant wasdiscarded. Cells were resuspended in 10 ml of fresh media. Cell densityand viability was determined by trypan blue.

For positive selection of anti-PACAP38 producing B-cells, biotinylatedPACAP38 (SEQ ID NO: 1241) was pre-loaded onto the streptavidin beads asfollows. 75 μl of streptavidin beads (Miltenyi Biotec, Auburn, Calif.)were mixed with N-terminally biotinylated PACAP38 (10 μg/ml finalconcentration) and 300 μl of PBS supplemented with 0.5% biotin freebovine serum albumin (“BSA”) and 2 mM EDTA (“PBF”). This mixture wasincubated at 4° C. for 30 minutes, and unbound biotinylated PACAP38(AnaSpec, Fremont, Calif.) was removed using a MACS® separation column(Miltenyi Biotec, Auburn, Calif.) with a 1 ml rinse to remove unboundmaterial. The bound material was plunged out by detachment from themagnet and used to resuspend cells from above in 100 μl per 1×10⁷ cells.The mixture was then incubated at 4° C. for 30 minutes and washed oncewith 10 ml of PBF. After washing, the cells were resuspended in 500 μlof PBF and set aside. A MACS® MS column (Miltenyi Biotec, Auburn,Calif.) was pre-rinsed with 500 μl of PBF on a magnetic stand (MiltenyiBiotec, Auburn, Calif.). Cell suspension was applied to the columnthrough a pre-filter, and unbound fraction was collected. The column waswashed with 2.5 ml of PBF buffer. The column was removed from the magnetstand and placed onto a clean, sterile 1.5 ml EPPENDORF™ tube. 1 ml ofPBF buffer was added to the top of the column, and positive selectedcells were collected. The yield and viability of positive cell fractionwas determined by trypan blue staining. Positive selection yielded anaverage of 1% of the starting cell concentration.

A pilot cell screen was established to provide information on seedinglevels for the culture. Plates were seeded at 5, 10, 25, 50, 100, or 200enriched B-cells/well. In addition, each well contained 25-50Kcells/well of irradiated EL-4.B5 cells (5,000 Rads) and an appropriatelevel of activated rabbit T-cell supernatant (See U.S. PatentApplication Publication No. 20070269868) (ranging from 1-5% depending onpreparation) in high glucose modified RPMI medium at a final volume of250 μl/well. Cultures were incubated for 5 to 7 days at 37° C. in 4%CO₂.

B-Cell Culture Screening by Antigen-Recognition (ELISA)

To identify wells producing anti-PACAP38 antibodies, B-cell supernatantswere tested by antigen-recognition (ELISA). Briefly, NEUTRAVIDIN™-coatedplates (Thermo Fisher Scientific, Waltham, Mass.), were coated witheither N-term or C-term biotinylated PACAP38 (AnaSpec Inc., Fremont,Calif.) (50 μl per well; 1 μg/ml) diluted in ELISA buffer (0.5% fishskin gelatin in PBS pH 7.4) either for approximately 1 hour at roomtemperature or alternatively overnight at 4° C. The plates were thenfurther blocked with ELISA buffer for one hour at room temperature andwashed using PBS with 0.05% Tween 20 (“wash buffer”). B-cell supernatantsamples (50 μl) were transferred onto the wells and incubated for onehour at room temperature. After this incubation, the plate was washedwith wash buffer. For development, an anti-rabbit specific Fc-HorseRadish Peroxidase (“Fc-HRP”) (1:5000 dilution in ELISA buffer) was addedonto the wells and incubated for 45 minutes at room temperature. After a3× wash step with wash solution, the plate was developed using3,3′,5,5′-Tetramethylbenzidine (“TMB”) substrate for two minutes at roomtemperature, and the reaction was quenched using 0.5M HCl. The wellabsorbance was read at 450 nm.

To identify wells producing anti-PACAP38 antibodies that do notrecognize VIP (SEQ ID NO: 1243), supernatant from wells positive forPACAP38 binding by ELISA were tested by ELISA for binding to VIP.Briefly, biotinylated VIP (AnaSpec Inc., Fremont, Calif.) was bound ontoNEUTRAVIDIN™ coated plates (50 μg per well, 1 μg/μl each peptide).B-cell supernatant samples (50 μl) were tested without prior dilution.Recognition in this assay may indicate cross reactivity with a closelyrelated peptide, VIP.

Identification of Functional Activity in B-Cell Supernatants Using Oneor More Assays

To identify wells producing anti-PACAP38 antibodies that block signalingof PACAP38 via PAC1-R, supernatant from positive wells for PACAP38binding by ELISA were tested in a cAMP HTRF assay (Cisbio US, Bedford,Mass.). Supernatants (78 μl) were pre-incubated with 2 μl nM PACAP38(American Peptide Company, Sunnyvale, Calif.) for 1 hour at 37° C.During the incubation, PC-12 cells were prepared as described for titerassessment. Cells (10 μl) and antigen/antibody complex (40 μl) weretransferred to an HTRF plate and shaken at room temperature for 30minutes. Following the incubation, 20 μl of (1:20 diluted) Eu³⁺cryptate-labeled mAb anti-cAMP and 20 μl of (1:20 diluted) d2-labeledcAMP in lysis buffer were added, and the plate was incubated for 1 hourwhile shaking. Following incubation plates were read (excitation 330 nm,emission 620/665 nm), and a ratio of 620:665 signal was determined.

Isolation of Antigen-Specific B-Cells

Antigen-specific B-cells were isolated (for general methods see co-ownedpublication no. WO 2014/146074, which is hereby incorporated byreference in its entirety). Plates containing wells of interest wereremoved from −70° C., and the cells from each well were recovered usingfive washes of 200 μl of medium (10% RPMI complete, 55 μM(3-mercaptoethanol (“BME”)) per well. The recovered cells were pelletedby centrifugation and the supernatant was carefully removed. Cells fromeach well were then re-suspended in 100 μl of medium and transferred toa 96 well plate. Cells were incubated for 90 minutes at 37° C. Followingincubation, cells were pelleted by centrifugation, stained with afluorescein isothiocyanate-labeled (“FITC-labeled”) anti-rabbit IgG(final concentration 6.25 μg/ml) (Creative Diagnostics, Shirley, N.Y.),and washed with up to 2 ml fluorescence-activated cell sorting buffer(“FACS buffer”) (Dulbecco's PBS w/2% FBS) and re-suspended in 250 μl ofFACS buffer.

Control wells from the same culture sets that were similar incomposition to pooled wells of interest were thawed and stainedalongside target wells. These samples were initially run on FACS (BDINFLUX™, Becton, Dickinson and Company, Franklin Lakes, N.J.), and gateswere established for IgG, viability, and physical parameters (Forwardscatter (“FSC”)/side scatter (“SSC”)) that differentiate B-cells fromthe murine EL4 cells. Once gates were established, the sample ofinterest was run, and IgG positive, viable cells that were of aconsistent physical (FSC/SSC) population were sorted individually intowells of a 96 well plate pre-loaded with RT-PCR master mix. Upwards of 8cells per well were sorted. Sorted plates were removed from the sorterand transferred directly to thermocyclers for PCR.

Amplification and Sequence Determination of Antibody Sequences fromFACS-Sorted B-Cells

Antibody sequences were recovered using a combined RT-PCR based methodfrom a single cell sorted B-cell. Primers containing restriction enzymeswere designed to anneal in conserved and constant regions of the targetimmunoglobulin genes (heavy and light), such as rabbit immunoglobulinsequences, and a two-step nested PCR recovery was used to amplify theantibody sequence. Amplicons from each well were sequenced and analyzed.Representative antibodies from the resulting sequence clusters wereselected for recombinant protein expression. The original heavy andlight variable regions amplified from rabbit cells were cloned intohuman heavy and light chain constant region expression vectors viarestriction enzyme digestion and ligation, and via Gibson method.Vectors containing subcloned DNA fragments were amplified and purified.The sequences of the subcloned heavy and light chains were verifiedprior to expression.

Recombinant Production of Monoclonal Antibody of Desired AntigenSpecificity and/or Functional Properties

To determine antigen specificity and functional properties of recoveredantibodies from specific B-cells, the heavy and light chain plasmidswere co-transfected to generate rabbit/human chimeric antibodies fortesting. Briefly, heavy and light chimeric plasmids were transientlytransfected into HEK-293 cells. Transfections were allowed to incubatefor 5-7 days, and upon harvest, cells were pelleted by centrifugation.Supernatants were submitted for purification via Protein A. Resultingpurified chimeric antibodies were then evaluated in a variety of assaysto confirm specificity and potency.

Using the above-described methods, numerous functional (antagonistic)antibodies that bind PACAP38 and PACAP27, or that bind PACAP38 only, butwhich do not, or do not appreciably, bind to VIP were identified.Polypeptide and exemplary coding sequences of exemplary antagonisticanti-PACAP antibodies are contained in the included biological sequencelisting.

The full-length antibodies Ab1, Ab1.H, Ab2, Ab13, Ab14, Ab15, Ab16,Ab17, Ab18, Ab19, Ab5, Ab7, Ab11, Ab12, Ab4, Ab3, Ab6, Ab8, and Ab9 usedin these examples were expressed as the heavy chain polypeptides havingthe sequences of SEQ ID NOS: 1; 41; 81; 121; 161; 201; 241; 281; 321;361; 481; 521; 561; 601; 641; 681; 721; 761; and 801, respectively, andthe light chain polypeptides of SEQ ID NOS: 21; 61; 101; 141; 181; 221;261; 301; 341; 381; 501; 541; 581; 621; 661; 701; 741; 781; 821,respectively. The heavy chain polypeptides of antibodies Ab1, Ab1.H,Ab2, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab5, Ab7, Ab11, Ab12,Ab4, Ab3, Ab6, Ab8, and Ab9 were expressed from the polynucleotides ofSEQ ID NOS: 11; 51; 91; 131; 171; 211; 251; 291; 331; 371; 491; 531;571; 611; 651; 691; 731; 771; 811, respectively. The light chainpolypeptides of antibodies Ab1, Ab1.H, Ab2, Ab13, Ab14, Ab15, Ab16,Ab17, Ab18, Ab19, Ab5, Ab7, Ab11, Ab12, Ab4, Ab3, Ab6, Ab8, and Ab9 wereexpressed from the polynucleotides of SEQ ID NOS: 31; 71; 111; 151; 191;231; 271; 311; 351 391; 511; 551; 591; 631; 671; 711; 751; 791; and 831,respectively. Additional features of said antibodies are identified bySEQ ID NOS in FIGS. 1-12.

Antigen Binding Specificity of Antibodies by Competitive HTRF BindingAssay

The binding and functional properties of exemplary anti-PACAP38 andanti-PACAP27 antibodies produced according to the invention are furtherdescribed below.

To identify antibodies that preferentially bind PACAP38 (SEQ ID NO:1241) and PACAP27 (SEQ ID NO: 1242), but do not bind VIP (SEQ ID NO:1243), or to identify antibodies that specifically bind PACAP38, but donot bind appreciably PACAP27, or do not appreciably bind VIP, etc., acompetition HTRF binding assay was performed.

In parallel, 10 μl of an antibody dilution series (highest finalconcentration of 100 nM) were incubated with 10 μl of N-terminal orC-terminal biotinylated PACAP38 (35 nM final) alone, or in combinationwith either PACAP27 (350 nM final) or VIP (350 nM final), i.e., 10×PACAP27 or 10×VIP, respectively, in a HTRF plate. 20 μl of Eu³⁺ cryptatelabeled anti-hu Fc donor and 20 μl of d2-labeled streptavidin acceptorwere added to each well and incubated for 1 hour at room temperature.Fluorescence was measured at 620 and 665 nm with a delay of 300 μsec.

Binding data were obtained based on experiments assaying the binding ofthe subject anti-PACAP antibodies to PACAP38 and to PACAP27. Thesebinding curve data showed that VIP is unable to effectively compete withbinding of PACAP38. The lack of effect of VIP on binding to PACAP38indicated its inability to compete with binding of PACAP38. Theseresults demonstrated that Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, and Ab1.Hbind to PACAP38 and PACAP27, but do not bind (or do not appreciablybind) VIP. These results also demonstrated that Ab22 and Ab23 bind toPACAP38, but do not bind (or do not appreciably bind) PACAP27 or VIP.

EC₅₀ values, i.e. the concentration of an antibody that yields aresponse halfway between the baseline and the maximum value within aspecified time period, were computed for each antibody based upon theirbinding curves and are shown in Table 1 below. The results demonstratedthat Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12,Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab22, Ab23, and Ab1.H bound toand recognized human PACAP38 with high affinity. A humanized form ofantibody Ab1 was produced and is identified by an appended “.H”, i.e.,Ab1.H also bound PACAP38 with high affinity.

TABLE 1 Binding (EC₅₀) of PACAP38 by anti-PACAP antibodiesPACAP38-binding ANTIBODY EC₅₀ ₍nM) Ab1 0.43 Ab2 0.35 Ab3 0.45 Ab4 0.66Ab5 0.60 Ab6 0.50 Ab7 0.45 Ab8 0.48 Ab9 0.23 Ab10 0.36 Ab11 0.53 Ab120.51 Ab13 0.48 Ab14 0.57 Ab15 0.62 Ab16 0.68 Ab17 0.46 Ab18 0.48 Ab190.43 Ab1.H 0.46 Ab22 0.57 Ab23 0.56Ability of Anti-PACAP Antibodies to Neutralize PACAP38-Induced andPACAP27-Induced cAMP Production

The ability of anti-PACAP antibodies to neutralize PACAP38-induced andPACAP27-induced PAC1-R signaling was tested in a cell-based assay.

For Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13,Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab22, Ab23, Ab1.H, Ab10.H, Ab3.H,Ab4.H, Ab5.H, Ab9.H and Ab12.H, to identify antibodies that neutralizedPACAP38-induced and PACAP27-induced signaling via PAC1-R, antibodysolutions were incubated with either PACAP38 or with PACAP27 at 4× thefinal concentration (100 pM) for 1 hour. While the antibody/antigencomplexes were incubated, PAC1-R expressing PC-12 cells (JapaneseCollection of Research Bioresources Cell Bank) were washed andre-suspended at 2×10⁶ cells per ml in cell culture media. Cells (10 μl)and antigen/antibody complex (40 μl) were transferred to an HTRF plateand shaken at room temperature for 30 minutes. Following the incubation,20 μl of (1:20 diluted) Eu³⁺ cryptate-labeled mAb anti-cAMP and 20 μl of(1:20 diluted) d2-labeled cAMP in lysis buffer were added, and the platewas incubated for 1 hour while shaking. Following incubation, plateswere read (excitation 330 nm, emission 620/665 nm), and a ratio of620:665 signal was determined. The final concentration of PACAP38 andPACAP27 in each well was 0.1 nM.

Inhibition curves were obtained for Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7,Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19,Ab1.H, Ab10.H, Ab22, Ab23, Ab3.H, Ab4.H, Ab5.H, Ab9.H, and Ab12.H. Theinhibition results were quantified for each antibody to yield an IC₅₀value, which are summarized in Table 2 below. These results demonstratedthat anti-PACAP antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab22, Ab23,Ab1.H, Ab10.H, Ab3.H, Ab4.H, Ab5.H, Ab9.H, and Ab12.H inhibitedPACAP38-induced cAMP increase in cells expressing PAC1-R. Additionally,these results demonstrated that anti-PACAP antibodies Ab1, Ab2, Ab3,Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16,Ab17, Ab18, Ab19, Ab1.H, Ab10.H, Ab3.H, Ab4.H, Ab5.H, Ab9.H, and Ab12.H,but not Ab22 or Ab23, inhibited PACAP27-induced cAMP increase in cellsexpressing PAC1-R.

TABLE 2 Inhibition (IC₅₀) of PACAP38-induced and PACAP27-induced cAMPincrease in cells expressing PAC1-R by anti-PACAP antibodies Inhibitionof 0.1 nM Inhibition of 0.1 nM PACAP38-induced PACAP27- PAC1-R mediatedinduced PAC1-R cAMP increase IC₅₀ mediated cAMP ANTIBODY ₍pM) increaseIC₅₀ ₍pM) Ab1 292.9 72.9 Ab2 236.6 63.0 Ab3 227.3 90.0 Ab4 340.4 76.9Ab5 326.9 353.3 Ab6 51.2 380.0 Ab7 111.3 45.8 Ab8 39.3 49.0 Ab9 987.02840.0 Ab10 180.3 227.0 Ab11 56.7 109.3 Ab12 51.1 60.4 Ab13 82.4 74.1Ab14 154.4 95.7 Ab15 162.0 155.5 Ab16 211.8 192.4 Ab17 97.7 77.6 Ab18117.7 91.6 Ab19 100.8 87.4 Ab1.H 259.6 57.7 Ab10.H 163.4 84.0 Ab22 101.4n/a* Ab23 114.9 n/a* Ab3.H 1320.0 1207.0 Ab4.H 307.0 293.6 Ab5.H 378.4216.8 Ab9.H 278.0 270.5 Ab12.H 113.9 86.3 *n/a: not active because theseAbs are PACAP38 specific

Example 2: Binding Affinities of Anti-PACAP Antibodies

Binding affinities of monoclonal antibodies for human PACAP wereestimated using SPR on the PROTEON™ XPR36 (Bio-Rad, Hercules, Calif.).Antibody was immobilized to the surface of general amine coupling (“GLC”or “GLM”) Chips (Bio-Rad, Hercules, Calif.). A dilution series of humanPACAP38 (SEQ ID NO: 1241) prepared in 1×PBST Buffer (4.3 mM NaPhosphate, 1.4 mM K Phosphate, 135 mM NaCl, 2.7 mM KCl 0.05%Polysorbate-20) purchased from Teknova (Cat# P1192, Teknova, Hollister,Calif.) and supplemented with 0.25 M arginine (from J.T. BAKER®), 0.2mg/ml BSA (Jackson Immuno Research Labs, West Grove, Pa.), and 0.005%sodium azide (VWR International, Radnor, Pa.) with the pH adjusted to 7was used to query the antibodies. Antigen (ranging from 1.23 nM to 100nM) was typically run sequentially with association times of 2-4 minutesand dissociation times of 3-120 minutes grouped with the PROTEON™Manager Software (v3.1.0.6 (Bio-Rad, Hercules, Calif.)) and fitted usinga 1:1 Langmuir binding model. Surfaces were regenerated between analytequeries using 0.85% Phosphoric Acid. A single K_(D) was calculated foreach antibody with association times limited near the rate of diffusion(1.0×10⁶) and dissociation times limited to 1.5×10⁻⁵ where nodiscernible dissociation was observed.

The same procedure was used to determine binding affinities ofantibodies for human VIP (SEQ ID NO: 1243) and PACAP27 (SEQ ID NO: 1242)though peptide concentrations ranged from 1.23 nM to 1000 nM withassociation times of 200 seconds and dissociation times of 3-120minutes.

The measured antibody affinities for PACAP38 are listed in Table 3.

TABLE 3 Antibody affinity constants for PACAP38 Antibody ka (1/Ms) kd(1/s) K_(D) (M) Ab1 3.7E+05 1.0E−05 2.7E−11 Ab2 2.3E+05 1.0E−05 4.4E−11Ab3 2.6E+05 4.2E−05 1.6E−10 Ab4 3.3E+05 7.2E−05 2.2E−10 Ab5 2.4E+051.0E−05 4.1E−11 Ab6 3.0E+05 4.1E−05 1.4E−10 Ab7 1.8E+05 1.0E−05 5.6E−11Ab8 3.5E+05 1.0E−05 2.9E−11 Ab9 8.2E+05 1.1E−04 1.4E−10 Ab10 2.6E+052.0E−05 7.5E−11 Ab11 2.7E+05 3.1E−05 1.1E−10 Ab12 3.1E+05 1.0E−053.2E−11 Ab13 4.2E+05 2.7E−05 6.4E−11 Ab14 3.2E+05 1.0E−05 3.1E−11 Ab155.3E+05 2.7E−05 5.0E−11 Ab16 9.1E+05 1.0E−05 1.1E−11 Ab17 5.0E+051.0E−05 2.0E−11 Ab18 4.3E+05 1.0E−05 2.3E−11 Ab19 2.7E+05 2.5E−059.3E−11 Ab22 3.7E+05 1.0E−05 2.7E−11 Ab23 5.1E+05 3.6E−05 7.1E−11 Ab1.H4.7E+05 1.0E−05 2.1E−11 Ab3.H 4.9E+05 1.4E−04 2.9E−10 Ab4.H 3.1E+053.2E−05 1.0E−10 Ab5.H 5.5E+05 1.7E−05 3.1E−11 Ab9.H 1.0E+06 6.1E−056.1E−11 Ab10.H 3.4E+05 1.0E−05 2.9E−11 Ab12.H 3.8E+05 1.0E−05 2.6E−11

Examples of antibody affinity constants for VIP are listed in Table 4.

TABLE 4 Antibody affinity constants for VIP Antibody ka (1/Ms) kd (1/s)K_(D) (M) Ab1 2.5E+05 2.5E−02 9.9E−08 Ab2 5.8E+05 8.4E−02 1.4E−07 Ab31.0E+00 1.0E−01 1.0E−01 Ab4 1.7E+05 2.1E−02 1.2E−07 Ab5 1.2E+05 8.6E−017.2E−06 Ab6 3.1E+03 1.4E−04 4.4E−08 Ab7 2.6E+05 8.8E−03 3.4E−08 Ab84.8E+05 1.0E−01 2.1E−07 Ab9 1.0E+00 1.0E−01 1.0E−01 Ab10 3.7E+04 1.0E−022.8E−07 Ab11 2.0E+05 4.7E−02 2.3E−07 Ab12 2.9E+05 2.4E−03 8.2E−09 Ab133.2E+05 4.6E−02 1.4E−07 Ab14 2.7E+05 6.7E−02 2.5E−07 Ab15 1.6E+051.3E−01 8.2E−07 Ab16 3.6E+05 9.6E−02 2.6E−07 Ab17 3.1E+05 1.7E−025.5E−08 Ab18 3.1E+05 1.2E−01 4.0E−07 Ab19 2.8E+05 2.8E−01 1.0E−06 Ab222.7E+05 1.8E−01 6.9E−07 Ab23 4.3E+05 3.2E−01 7.3E−07 Ab1.H 3.8E+041.8E−01 4.8E−06 Ab3.H 1.0E+00 1.0E−01 1.0E−01 Ab4.H 3.3E+05 2.4E−027.2E−08 Ab5.H 1.0E+00 1.0E−01 1.0E−01 Ab9.H 9.3E+04 1.4E−01 1.5E−06Ab10.H 3.8E+05 3.9E−02 1.0E−07 Ab12.H 2.8E+05 1.4E−02 5.1E−08

Examples of antibody affinity constants for PACAP27 are listed in Table5.

TABLE 5 Antibody affinity constants for PACAP27 Antibody ka (1/Ms) kd(1/s) K_(D) (M) Ab1 1.0E+06 1.0E−05 1.0E−11 Ab2 8.3E+05 1.0E−05 1.2E−11Ab3 3.7E+05 1.4E−04 3.7E−10 Ab4 3.9E+05 2.0E−04 5.1E−10 Ab5 2.5E+052.4E−05 9.6E−11 Ab6 3.9E+05 8.2E−05 2.1E−10 Ab7 2.3E+05 4.5E−05 2.0E−10Ab8 4.4E+05 6.0E−05 1.4E−10 Ab9 9.6E+05 3.2E−04 3.4E−10 Ab10 1.0E+061.0E−05 1.0E−11 Ab11 2.6E+05 1.1E−04 4.2E−10 Ab12 2.9E+05 2.1E−057.0E−11 Ab13 5.4E+05 6.6E−05 1.2E−10 Ab14 2.7E+05 2.0E−05 7.4E−11 Ab154.7E+05 7.8E−05 1.7E−10 Ab16 8.2E+05 2.9E−05 3.5E−11 Ab17 3.7E+051.0E−05 2.7E−11 Ab18 4.4E+05 1.0E−05 2.3E−11 Ab19 4.2E+05 1.3E−043.1E−10 Ab22 1.0E+00 1.0E−01 1.0E−01 Ab23 8.9E+05 3.1E−02 3.5E−08 Ab1.H7.6E+05 1.0E−05 1.3E−11 Ab3.H 3.3E+05 3.4E−04 1.0E−09 Ab4.H 3.3E+051.0E−05 3.1E−11 Ab5.H 2.7E+05 1.3E−04 4.8E−10 Ab9.H 6.2E+05 7.7E−051.2E−10 Ab10.H 5.3E+05 1.8E−05 3.3E−11 Ab12.H 2.6E+05 7.9E−05 3.0E−10

The binding affinity results of Tables 3 and 5 present datademonstrating that Ab23 weakly bound to PACAP27 as compared to itsbinding affinity for PACAP38. Tables 3 and 5 additionally present datademonstrating that Ab22 did not specifically recognize PACAP27, but thatAb22 specifically bound to PACAP38.

Example 3: Inhibition of PACAP38-Induced Signaling Via VPAC1-R

To identify antibodies that neutralize PACAP38-induced signaling viahuman VPAC1-R, CHO-K1 cells expressing human VPAC1-R were used in a cAMPHTRF cell-based assay. Antibody dilutions were incubated with PACAP38 at4× the final concentration (5 nM) for 1 hour. While the antibody/antigencomplexes were incubated for 1 hour, VPAC1-R expressing CHO-K1 cells(generated at Alder Biopharmaceuticals, by stable transfection of CHO-K1cells (ATCC, catalog # CCL-61) with human VPAC1-R cDNA; selected clone 1was used for in vitro cell based assays) were detached with 0.25%trypsin for 4 minutes. The cells were washed and re-suspended at 1×10⁶cells per ml culture media. 20 μl of Ab/antigen mixture was mixed with20 μl of cells in HTRF plates and incubated with shaking for 30 minutes.20 μl of Eu³⁺ cryptate labeled anti-cAMP mAb (1:20 diluted) and 20 μl of(1:20 diluted) d2-labeled cAMP in lysis buffer were added to each welland incubated for 1 hour with shaking. The final concentration ofPACAP38 in each well was 5 nM. Following incubation, plates were read(excitation 330 nm, emission 620/665 nm), and a ratio of 620:665 signalwas determined.

Inhibition curves were obtained for Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7,Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19,Ab1.H, Ab10.H, Ab22, Ab23, Ab3.H, Ab4.H, Ab5.H, Ab9.H, and Ab12.H. Thecomputed IC₅₀ values for each antibody, which are shown below in Table6, demonstrated that Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10,Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab1.H, Ab10.H,Ab22, Ab23, Ab3.H, Ab4.H, Ab5.H, Ab9.H, and Ab12.H inhibitedPACAP38-induced cAMP increase in cells expressing human VPAC1-R.

TABLE 6 Inhibition (IC₅₀) of PACAP38-induced cAMP increase in cellsexpressing human VPAC1-R by anti-PACAP antibodies Inhibition of 5 nMPACAP38-induced Human VPAC1-R mediated cAMP ANTIBODY increase IC₅₀ ₍pM)Ab1 664.7 Ab2 688.3 Ab3 1736.0 Ab4 942.8 Ab5 720.7 Ab6 797.1 Ab7 687.3Ab8 481.2 Ab9 4059.0 Ab10 649.1 Ab11 541.0 Ab12 292.2 Ab13 2183.0 Ab142626.0 Ab15 3715.0 Ab16 3533.0 Ab17 780.1 Ab18 911.2 Ab19 826.8 Ab1.H1021.1 Ab10.H 1336.0 Ab22 1300.0 Ab23 2667.0 Ab3.H 7332.0 Ab4.H 2600.0Ab5.H 2772.0 Ab9.H 2465.0 Ab12.H 1284.0

Example 4: Inhibition of PACAP38-Induced Signaling Via VPAC2-R

To identify antibodies that neutralize PACAP38-induced signaling viahuman VPAC2-R, CHO-K1 cells expressing human VPAC2-R were used in a cAMPHTRF cell based assay. Antibody dilutions were incubated with PACAP38 at4× the final concentration (1 nM) for 1 hr. While the antibody/antigencomplexes were incubated for 1 hour, VPAC2-R expressing CHO-K1 cells(generated at Alder Biopharmaceuticals, by stable transfection of CHO-K1cells (ATCC, catalog # CCL-61) with human VPAC2-R cDNA; selected clone 8was used for in vitro cell based assays) were detached with 0.25%trypsin for 4 minutes. The cells were washed and re-suspended at 1×10⁶cells per ml culture media. 20 μl of Ab/antigen mixture was mixed with20 μl of cells in HTRF plates and incubated with shaking for 30 minutes.20 μl of Eu³⁺ cryptate labeled anti-cAMP mAb (1:20 diluted) and 20 μl of(1:20 diluted) d2-labeled cAMP in lysis buffer were added to each welland incubated for 1 hour with shaking. The final concentration ofPACAP38 in the wells was 1 nM. Following incubation, plates were read(excitation 330 nm, emission 620/665 nm) and, a ratio of 620:665 signalwas determined.

Inhibition curves were obtained using this method for Ab1, Ab2, Ab3,Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16,Ab17, Ab18, Ab19, Ab1.H, Ab10.H, Ab22, Ab23, Ab3.H, Ab4.H, Ab5.H, Ab9.H,and Ab12.H. The computed IC₅₀ values for each antibody, which are shownbelow in Table 7, demonstrated that Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7,Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19,Ab1.H, Ab10.H, Ab22, Ab23, Ab3.H, Ab4.H, Ab5.H, Ab9.H, and Ab12.Hinhibited PACAP38-induced cAMP increase in cells expressing humanVPAC2-R.

TABLE 7 Inhibition (IC₅₀) of PACAP38-induced cAMP increase in cellsexpressing human VPAC2-R by anti-PACAP antibodies Inhibition of 1 nMPACAP38- induced human VPAC2-R mediated cAMP increase IC₅₀ ANTIBODY (pM)Ab1 146.7 Ab2 174.1 Ab3 667.4 Ab4 217.9 Ab5 239.3 Ab6 216.9 Ab7 162.4Ab8 146.9 Ab9 6965.0 Ab10 188.5 Ab11 265.2 Ab12 179.0 Ab13 652.2 Ab14840.4 Ab15 22850.0 Ab16 1146.0 Ab17 205.0 Ab18 285.4 Ab19 953.5 Ab1.H983.0 Ab10.H 988.0 Ab22 515.0 Ab23 1789.0 Ab3.H 64240.0 Ab4.H 4487.0Ab5.H 7466.0 Ab9.H 2649.0 Ab12.H 653.0

Example 5: Inhibition of PACAP38 Binding to PAC1-R-Expressing Cells

To identify antibodies that block PACAP38 binding to PAC1-R-expressingcells, adherent PC-12 cells (ATCC, Manassas, Va.) expressing PAC1-R wereused in a Europium-based PAC1-R-expressing cells binding assay.

Antibody solutions were incubated with N-terminal biotinylated PACAP38at 10× the final concentration (100 nM or 30 nM) for 1 hr, then added toPC-12 cells that were plated 24 hrs prior in black clear bottom 96 wellplates (COSTAR™, Corning Incorporated, Corning, N.Y.) and furtherincubated for 1 hr at room temperature. After three washes, the cellswere incubated with 20 μl Europium-labeled streptavidin (PerkinElmer,Waltham, Mass.) for 1 hr at room temperature. Cells were washed threetimes, then 20 μl DELFIA® Enhancement solution (PerkinElmer, Waltham,Mass.) was added to each well and incubated for 15 minutes with gentleshaking. Plates were read (Time Resolved Fluorescence (“TRF”)) onSPECTRAMAX® (Molecular Devices, Sunnyvale, Calif.) plate reader.

Inhibition curves were obtained using this method for Ab1, Ab2, Ab3,Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16,Ab17, Ab18, Ab19, Ab1.H, Ab10.H, Ab22, Ab23, Ab3.H, Ab4.H, Ab5.H, Ab9.H,and Ab12.H wherein the PAC1-R expressing cells were PC-12 cells. Thecomputed IC₅₀ values for each antibody, which are shown below in Table8, demonstrated that Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10,Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab1.H, Ab10.H,Ab22, Ab23, Ab3.H, Ab4.H, Ab5.H, Ab9.H, and Ab12.H inhibited PACAP38binding to PAC1-R expressing cells.

TABLE 8 Inhibition (IC₅₀) of PACAP38 binding to PAC1-R-expressing PC-12cells by anti-PACAP antibodies. ANTIBODY Inhibition of 100 nMbiotinylated PACAP38 binding to PAC1R-expressing PC-12 cells IC₅₀ (nM)Ab1 12.7 Ab2 26.6 Ab3 17.2 Ab4 28.6 Ab5 16.3 Ab6 7.8 Ab7 22.0 Ab8 20.3Ab9 33.3 Ab10 17.8 Ab11 24.6 Ab12 22.6 Ab13 53.1 Ab14 17.9 Ab15 36.7Ab16 27.3 Ab17 33.1 Ab18 30.6 Ab19 162 Inhibition of 30 nM BiotinylatedPACAP38 binding to PAC1R-expressing PC-12 cells IC₅₀ (nM) Ab1.H 56.3Ab10.H 14.5 Ab22 13.8 Ab23 14.9 Ab3.H 88.3 Ab4.H 98.0 Ab5.H 34.9 Ab9.H22.5 Ab12.H 68.1

Example 6: PACAP38-Mediated Binding of Anti-PACAP Antibodies to the CellSurface of PAC1-R Expressing Cells

To identify anti-PACAP antibodies that bind, via PACAP38, to the cellsurface of PAC1-R expressing cells, adherent PC-12 cells (JapaneseCollection of Research Bioresources Cell Bank) expressing PAC1-R wereused in a cell surface binding-based assay. To perform the bindingexperiment, PAC1-R expressing PC-12 cells were first seeded into Corning96 well white solid bottom plates (Corning, Corning, N.Y.). Cells wereinitially seeded at 1×10⁵ cells/well in a solution of complete RPMI(“cRPMF”: RPMI medium supplemented with 10% sterile heat-inactivated FBSand 1% sterile antibiotic/antimycotic)+10% FBS, and the plates wereallowed to incubate overnight at 37° C. On the day of the binding assay,antibodies at an initial concentration of 15 μg/ml were diluted at a 1:3ratio in DELFIA® binding buffer (50 mM Tris, 150 mM NaCl, 0.1% azide, 2%horse serum) (Perkin-Elmer, Waltham, Mass.) to a total volume of 60 μLin a separate 96 well round bottom plate. PACAP38 was prepared for thebinding assay by diluting it in DELFIA® binding buffer to aconcentration of 200 nM, and then 60 μl of the diluted PACAP38 was addedto each of the antibody-containing wells to form antibody:antigencomplexes. Following addition of PACAP38, the antibody:antigen complexeswere incubated at room temperature on a shaker for 1 hour. Separately,the PC-12 cells were prepared for addition of antibody:antigen complexesby washing the cells two times with DELFIA® wash buffer (50 mM Tris, 150mM NaCl, 0.1% Azide) (Perkin-Elmer, Waltham, Mass.). After washing thecells two times and following the 1 hour room temperature incubation ofthe antibody:antigen complexes, 50 μl of the antibody:antigen complexwas added to each well containing cells. The mixtures of cells andantibody:antigen complexes were then incubated for 30 minutes at roomtemperature. Following this 30 minute incubation, each mixture waswashed two times with DELFIA® wash buffer (Perkin-Elmer, Waltham,Mass.).

DELFIA® Europium labeled anti-human IgG detection reagent (Cat#1244-330, Perkin-Elmer, Waltham, Mass.) was diluted to a concentrationof 300 ng/ml in DELFIA® Binding Buffer. Following dilution, 50 μl of theanti-human IgG detection reagent was added to each well containingcells, and a 30 minute incubation at room temperature followed thisaddition of IgG detection reagent. After completion of the 30 minuteroom temperature incubation, the cells were then washed two times withDELFIA® wash buffer. Next, 50 μl of DELFIA® Enhancement Solution (Cat#1244-105, Perkin-Elmer, Waltham, Mass.) was added to each wellcontaining cells for a final 15 minute room temperature incubation withshaking. The plates were then read (TRF, excitation 330 nm, emission 620nm) on a SPECTRAMAX® (Molecular Devices, Sunnyvale, Calif.) platereader.

FIGS. 19A-J are representative of the binding curves obtained by thismethod (results are shown for Ab1.H, Ab3.H, Ab4.H, Ab5.H, Ab9.H, Ab12.H,Ab10, Ab10.H, Ab22, and Ab23, respectively) wherein the PAC1-Rexpressing cells were PC-12 cells. Ab1.H, Ab3.H, Ab4.H, Ab5.H, Ab9.H,and Ab12.H demonstrated binding to the surface of PAC1-R expressingcells in the presence of PACAP38, while Ab10, Ab10.H, Ab22, and Ab23 didnot appear to appreciably bind to the surface of PAC1-R expressing cellsusing this assay. The binding of Ab1.H, Ab3.H, Ab4.H, Ab5.H, Ab9.H, andAb12.H to the cell surface of PAC1-R cells was only observed in thepresence of PACAP38. Without intent to be bound by theory, it ishypothesized that the binding of the antibodies to the cell surface wasmediated by binding of PACAP38 to GAGs that were present on the cellsurface, since binding of PACAP38 by GAGs has been previouslydemonstrated as a PAC1-R receptor independent mechanism of PACAP38binding and internalization by PC-12 cells (see Doan et al. (2012),Juhàsz et al. (2014), and Neree et al. (2015)).

Example 7: Inhibition of PACAP38-Induced Dermal Vasodilation in Rabbitsby Anti-PACAP Antibody Ab1.H

Intradermal injection of PACAP38 has been shown to elicit a localizedvasodilation in rabbits and humans (Warren et al., J. Cardio.Pharmacol., 29(1): 83-87 (1992); Seelinger et al., Am. J. Path.,177(5):2563-2575 (2010)). An in vivo efficacy study was conducted todetermine the activity of Ab1.H to inhibit a localized dermalvasodilation induced by an intradermal injection of PACAP38 in male NewZealand White rabbits.

Groups of 4 rabbits were dosed with either 90 mg/kg of Ab1.H or withnegative control vehicle (25 mM histidine, 250 mM sorbitol, pH 6.0).Injections were performed by IV (ear vein) bolus administration on day0. Prior to each rabbit PACAP38 challenge, the scapular region of eachanimal was clipped free of hair and wiped with 20% (v/v) alcohol inwater. On day 2, the animals were pre-anesthetized with ketaminehydrochloride and maintained under deep anesthesia with isoflurane gas.Four sites (Region of Interest (“ROI”)) for injection were identified onthe back of each animal using a SHARPIE® permanent marker. Dermalvasodilation and blood perfusion were monitored using the PeriCam PSI NRsystem for Laser Speckle Contrast Analysis (“LASCA”) imaging (Perimed,Järfälla, Sweden), before (baseline) and for 35 minutes afterintradermal PACAP38 challenge. Intradermal PACAP38 challenge wasperformed as follows: each animal received single intradermaladministrations (100 μl/site) of vehicle (one site or ROI) and PACAP38at 30 pmoles/site (3 sites or 3 ROIs). The blood perfusion rates foreach ROI were reported by the PeriCam PSI NR system in Perfusion units(“PU”) and analyzed using PIMSoft (Ver. 1.5 (Perimed, Järfälla,Sweden)).

For each treatment group, the relative % PU change following Ab1.H ornegative control administration compared to baseline was calculated foreach ROI (% PU change for each PACAP38 challenge site−% PU change forthe vehicle site). The relative % PU change in the Ab1.H group wascompared to the relative % PU change in the Negative control group byperforming a two-tailed unpaired t-test statistical evaluation usingGraphPad Prism (version 5.0d, GraphPad Software, La Jolla, Calif.)software.

FIG. 13 demonstrates that Ab1.H inhibited PACAP38-induced dermalvasodilation in rabbits, indicating effectiveness of the antibody atneutralizing PACAP38 activity in vivo.

Example 8: Inhibition of PACAP38-Induced Dermal Vasodilation in Rabbitsby Anti-PACAP Antibody Ab10

Intradermal injection of PACAP38 has been shown to elicit a localizedvasodilation in rabbits and humans (Warren et al. (1992); Seelinger etal. (2010)). An in vivo efficacy study was conducted to determine theactivity of Ab10 to inhibit a localized dermal vasodilation induced byan intradermal injection of PACAP38 in male New Zealand White rabbits.

Groups of 4 rabbits were dosed with either 72 mg/kg of Ab10 or withisotype antibody control. Injections were by (ear vein) bolusintravenous administration on day 0. Prior to each rabbit PACAP38challenge, the scapular region of each animal was clipped free of hairand wiped with 20% (v/v) alcohol in water. On day 2, the animals werepre-anesthetized with ketamine hydrochloride and maintained under deepanesthesia with isoflurane gas. Four sites (ROIs) for injection wereidentified on the back of each animal using a SHARPIE® permanent marker.Dermal vasodilation and blood perfusion were monitored using the PeriCamPSI NR system for LASCA imaging (Perimed, Järfälla, Sweden), before(baseline) and for 35 minutes after intradermal PACAP38 challenge.Intradermal PACAP38 challenge was performed as follows: each animalreceived single intradermal administrations (100 μl/site) of vehicle(one site or ROI) and PACAP38 at 30 pmoles/site (3 sites or 3 ROIs). Theblood perfusion rates for each ROI were reported by the PeriCam PSI NRsystem in PU and analyzed using PIMSoft (Ver. 1.5 (Perimed, Järfälla,Sweden)).

For each treatment group, the relative % PU change following Ab10 orIsotype Ab control administration compared to baseline was calculatedfor each ROI (% PU change for each PACAP38 challenge site−% PU changefor the vehicle site). The relative % PU change in the Ab10 group wascompared to the relative % PU change in the Isotype Ab control group byperforming a two-tailed unpaired t-test statistical evaluation usingGraphPad Prism (version 5.0d, GraphPad Software, La Jolla, Calif.)software.

FIG. 14 demonstrates that Ab10 inhibited PACAP38-induced dermalvasodilation in rabbits, indicating effectiveness of the antibody atneutralizing PACAP38 activity in vivo.

Example 9: Epitope Binning of Anti-PACAP Antibodies, Ab1 and Ab10

Ab1 was biotinylated at a 10:1 molar ratio with biotin (Thermo FisherScientific, Waltham, Mass.) per manufacturer guidelines. A 5 stepbiolayer interferometry experiment was performed as follows: In step 1,streptavidin biosensors (Pall ForteBio LLC, Menlo Park, Calif.) wereequilibrated for 50 seconds in 1× kinetics buffer (a 1:10 dilution inDBS of Pall ForteBio LLC, Menlo Park, Calif., cat#18-5032). In step 2, a2 μg/ml dilution of biotinylated antibody Ab1 in 1× kinetics buffer wasimmobilized for 500 seconds onto Streptavidin biosensors. In step 3, theantibody-functionalized biosensors were incubated in a solution of 2 μMunlabeled PACAP peptide (American Peptide Company, Sunnyvale, Calif.,catalog #34-0-20) in 1× kinetics buffer for 200 seconds. In step 4, thesensors were placed into 67 nM solutions of either unlabeled antibodyAb10 (FIG. 15A) or unlabeled antibody Ab1 as control (FIG. 15B) in 1×kinetics buffer for a 1000 second association step. Stability of bindingwas monitored during step 5 for a 1000 second dissociation in 1×kinetics buffer. In FIG. 15A, the “sandwich-style” capture of Ab10 viaAb1-captured PACAP indicates simultaneous and non-competitive binding ofthese two antibodies to PACAP. The control experiment in FIG. 15B showsminimal “sandwich-style” capture of Ab1 via Ab1-captured PACAP. Theexperiment was conducted on a ForteBio OCTET® QK instrument (PallForteBio LLC, Menlo Park, Calif.) at 30° C. and 1000 RPM.

Example 10: Inhibition of PACAP27 Binding to Human PAC1-R by Anti-PACAPAntibodies

To identify antibodies that block PACAP27 binding to PAC1-R, antibodiesat an initial concentration of 30 nM were diluted in incubation buffer(50 mM Hepes pH 7.4, 1 mM CaCl₂, 5 mM MgCl₂, 0.2% BSA) and serial 1:3dilutions were performed. Antibody dilutions (30 nM, 10 nM, 3 nM, 1 nM,0.3 nM, 0.1 nM, 0.03 nM, 0.01 nM, 0.003 nM and 0.001 nM) were then mixedand pre-incubated at 25° C. for 30 minutes with 0.1 nM of ¹²⁵I-labelledPACAP27 in incubation buffer. The antibody: ¹²⁵I-labelled PACAP27mixture was then added to 0.5 μg aliquots of cell membranes derived fromChem-1 cells expressing human recombinant PAC1-R long isoform inincubation buffer. The mixture was then incubated for 1 hour at 25° C.Following incubation, the samples were filtered and washed. Afterward,the filters were counted to quantitate ¹²⁵I-labelled PACAP27. As anexperimental control, non-specific binding to the cell membranes wasestimated using 0.1 μM of labeled PACAP27. The results indicated thatAb1.H, Ab10.H, and Ab12.H were capable of blocking PACAP27 binding toPAC1-R, thereby demonstrating inhibition of ligand-receptor binding bythe tested antibodies presented in Table 9.

TABLE 9 Inhibition (IC₅₀) of 0.1 nM ¹²⁵I-PACAP27 binding to PAC1-R byanti-PACAP antibodies ANTIBODY IC₅₀ ₍nM) Ab1.H 0.70 Ab10.H 0.22 Ab12.H0.16

Example 11: Effect of Anti-PACAP Antibody on Light Aversion

To examine the effect of anti-PACAP antibodies on photophobia, a mousemodel was employed in which mice were administered PACAP to triggerphotophobia. Photophobia was detected using a light aversion assay usinga light-dark box as described in Kaiser et al., J. Neurosci.,32(44):15439-15449, 2012. Mice were then administered anti-PACAPantibodies Ab1.H or Ab10.H or an unrelated control antibody and theiraversion to light quantitated. Results are reflected in FIGS. 16-18.

Light Aversion Assay

As described in Kaiser et al., the testing chambers were a plexiglasopen field (27 cm wide×27 cm deep×20.3 cm high) containing three sets of16 beam infrared arrays (two sets of perpendicular beams cross at aheight of 1.0 cm to detect mouse location and locomotion, and the thirdbeam crosses the width of the chamber at a height of 7.3 cm to detectvertical activity). The field was divided in two equal sized zones by adark insert, which is a five-sided, black-colored plexiglas box with atop, but no floor. The use of infrared light beams allowed tracking inboth zones. An opening (5.2 cm×6.8 cm) in the dark insert allowed freemovement between zones. While the dark insert blocked direct light, somelight could still enter through the opening. Each testing chamber waslocated inside a sound-attenuating cubicle (56 cm wide×38 cm deep×36 cmhigh) with a fan for ventilation (Med Associates, Inc.®, St. Albans,Vt.). A computer using Activity Monitor v6.02 (Med Associated Inc.) wasused for recording data from the six chambers.

For each chamber, a LED panel was attached to the ceiling of thesound-attenuating cubicle. The LED panel contains 36 collimated 1 wattLEDs (5500k Daylight White) (LEDwholesalers.com, Burlingame, Calif.). Tocontrol light intensity, each LED panel was connected to a dimmable LEDdriver (LINEARdrive®; eldoLED America Inc., San Jose, Calif.) leading toa potential range of light intensity from 3.0×10² to 2.7×10⁴ lx. Levelswere further attenuated to 5.5×10¹ lx using wax paper placed on a clearplexiglass tray below the LEDs. Light intensity was measured withTraceable Dual-Display Light Meter (Control Company, Friendswood, Tex.)placed on the floor of the testing chamber. At 2.7×10⁴ lx, LED lightsgenerated some heat in the sound attenuating chamber with the dark zoneat ˜25° C. and light zone at ˜27° C.

On the day of the experiment, mice were transported from animal housingand allowed to acclimate to the testing room (˜22° C.) for at least 30to 60 minutes with standard overhead fluorescent lighting (˜200 lxinside the housing cage). Room lights remained on, unless notedotherwise. In addition, all sound-generating equipment were turned onduring acclimation and remained on until testing was complete. There wasminimal human presence in the room during acclimation. Behavioraltesting was performed between 0800 CST and 1400 CST. Any abnormalphysical conditions (e.g. missing eye) were noted.

Ten week old male and female CD1 mice were used in the study (strain#022, Charles River, Wilmington, Mass., US). Mice were allowed torecover from shipping for one to two weeks prior to testing.

Acclimation

All mice were acclimated in the testing room at least 30 to 60 minutesprior to being placed in the light/dark chamber. The light intensity inthe chamber was initially set to 2.7×10³ lx. The mice were tested forthirty minutes in the chamber every day they were exposed to thelight/dark chamber. Baseline time in light for each mouse was obtainedby exposing the mice to the light/dark chamber twice, with a period ofrest of three days between baseline measurements (FIGS. 16 and 18,“Baseline1” and “Baseline2,” or “Baseline”, respectively).

Treatment

The mice were administered 30 mg/kg of either anti-PACAP antibody orcontrol IgG antibody (negative control antibody having the sameframework as the tested antibodies and that recognizes digoxigenin) byi.p. injection. The mice were then returned to their home cage to restfor one day (24 hours) prior to testing. The mice were then administered0.6 mg/kg PACAP or vehicle by i.p. injection and rested for 30 minutes.The mice were then placed in the light/dark chamber for 30 minutes (FIG.16 and FIG. 18, “Treatment”). After each mouse was exposed to thelight/dark chamber, the light/dark chamber and components were cleanedwith germicidal wipes and dried. About 5 to 7 minutes after a mouse wasplaced in the light/dark chamber, the next mouse to be tested wasinjected with PACAP or vehicle, as described above. This interval wasapproximately the amount of time required to clean the light/darkchamber between experiments.

Motility Measurements

Motility was measured at 5 minute intervals over the 30 minute testingperiod as described in Kaiser et al., J. Neurosci., 2012. Briefly, thenumber of vertical movements, such as rearing, ambulatory distance (cm,the total distance traveled during ambulatory movement status),transitions, and resting (percentage of time spent breaking no newbeams), were measured by light beam. All motility parameters werenormalized to the time spent in each zone to account for differentamount of time spent in that zone; thus, the raw value for eachparameter was divided by the time spent in that zone during the 5 mininterval. Time spent in each chamber was analyzed using GraphPad Prismsoftware (GraphPad Software, San Diego, Calif.), and reported asmean±standard error of the mean (“SEM”). Comparison was calculated bytwo-way repeated measure ANOVA, with Bonferroni's multiple-comparisontest for post-hoc analysis.

Mice were excluded based on three criteria: (1) after the first twoexposures to the box the baseline time in light was analyzed and anymouse that spent +/−one standard deviation of mean time in light atbaseline was removed from the experiment and not given drug treatment,(2) mice were excluded from analysis if they were identified asstatistical outliers (box plot, 10-90%), and (3) mice were excluded ifthey moved less than 10% of the time (combined light and dark).

In two experiments comparing the response of mice administered eitherantibody Ab1.H or Ab10.H to control IgG, the results indicate that miceadministered either PACAP antibody Ab1.H or Ab10.H spent more time inlight as compared to IgG control mice. FIG. 16 shows that mice behavednormally and similarly in both baseline measurements. On the other hand,the data provided in FIG. 16 show that mice treated with control IgGantibody and then PACAP spent statistically less time in light (squares)than mice administered anti-PACAP antibody Ab1.H and then PACAP(circles). (See, FIG. 16, “Treatment”). The data provided in FIG. 18also show that mice behaved normally and similarly in baselinemeasurements. On the other hand, the data provided in FIG. 18 show thatmice treated with control IgG antibody and then PACAP spentstatistically less time in light (triangles) than mice administeredanti-PACAP antibody Ab10.H and then PACAP (inverted triangles). (See,FIG. 18, “Treatment”). Time between each measurement was three days. Themean±SEM is provided for each 5-minute interval. Mice administeredvehicle only behaved as normal controls. Data provided in FIG. 17 showsthat administration of either anti-PACAP antibody Ab1.H, or control IgG,and vehicle (“Veh+PAC Ab” and “Veh+Con Ab,” respectively) did notmarkedly alter mouse behavior. FIG. 17 also shows that the average timeof the mouse in light decreased when PACAP and control IgG wereadministered (“PACAP+Con Ab”), whereas mice administered anti-PACAPantibody Ab1.H and PACAP exhibited normal, non-light-sensitive behavior(“PACAP+PAC Ab”).

Example 12: Epitope Mapping of Anti-PACAP Antibodies

In order to determine the epitopes contained within PACAP to which theanti-PACAP antibodies and antigen binding fragments thereof of theinvention bind, alanine scanning experiments were used. To perform theseexperiments, PACAP peptides were synthesized with a single pointmutation in each position replacing the native amino acid with anAlanine (“Ala”), and the consequences of a single point mutation as itrelates to binding affinity of PACAP and an antibody were measured.

Since an alanine residue already occupies positions 18, 24, and 25 ofwild-type PACAP, according to convention, these Ala residues werereplaced with Valine (“Val”) to determine the possible effects of theremoval of the alanine at these positions on the binding of the subjectanti-PACAP antibodies to PACAP. Per the usual convention these Alamutants were labeled according to the position in PACAP 1-38 followed bythe letter code for the substituted amino acid, e.g., 10A indicatesPACAP 1-38 substituted with alanine at amino acid position 10. Bindingof monoclonal antibodies for human PACAP and each mutant peptide wasdetected using SPR on the PROTEON™ XRP36 (Bio-Rad Laboratories,Hercules, Calif.). Samples and sample controls were immobilized onto aPROTEON™ GLC sensor chip (Bio-Rad Laboratories, Hercules, Calif.) at asingle density using standard amine coupling. The running buffer usedfor immobilization was DPBS/modified (HYCLONE™, GE Healthcare LifeSciences, Marlborough, Mass.) and immobilization was conducted at 25° C.The PROTEON™ GLC sensor chip (Bio-Rad Laboratories, Hercules, Calif.)was initialized and pre-conditioned per the manufacturer's protocol(bi-directional injections of 0.5% SDS, 50 mM NaOH, 100 mM HCl).

The immobilization process was performed step-wise to ensure a uniqueantibody on the spots of the PROTEON™ Chip (Bio-Rad Laboratories,Hercules, Calif.). The surface of the chip was activated with a 1:1mixture of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide(“EDAC/NHS”) and flow rate of 30 μL/min×5 minutes. Antibody samples werepreviously dialyzed or exchanged to 10 mM HEPES 150 mM NaCl pH 7.2, andthe antibody concentration was quantified using a NANODROP™ 2000spectrophotometer (Thermo Fisher Scientific, Waltham, Mass.). Theimmobilization targeted 2000-3000 response units (“RU”). Antibodysamples (5 μg/ml) in 10 mM sodium acetate, pH 5.5, were flowed at 30×4minutes. Deactivation was achieved at a flow rate of 30 μL/min for 5minutes using 0.3 M ethanolamine concomitantly with the next activation.

Following immobilization, the running buffer was changed to 1×PBST (4.3mM sodium phosphate, 1.4 mM potassium phosphate, 135 mM NaCl, 2.7 mMKCl, 0.05% TWEEN®) with 0.2 M arginine HCl (to reduce non-specificbinding), BSA (0.2 mg/ml, as a carrier) and PROCLIN300® (0.005% as apreservative, Sigma Aldrich, St. Louis, Mo.) and the chip surface wasallowed to re-equilibrate with an injection of new running buffer. Stocksolutions of human PACAP peptide (1-38) and alanine/valine mutantpeptides (Molecular Weight(s): 4.5 kD) at a concentration of 1 mg/mlwere added to the running buffer to final concentrations of 0.45 μg/ml(100 nM). These mixtures were then used to query individual spots on thechip surface with flow rates of 100 μL/min×2 minutes and allowed todissociate for 600 seconds. Chip surfaces were regenerated betweenanalytes by the addition of 0.85% phosphoric acid.

Each of antibodies Ab1, Ab2, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19,Ab5, Ab7, Ab11, Ab12, Ab4, Ab3, Ab6, Ab8, Ab9, Ab22, and Ab23 wereexamined under the same conditions as herein described

Sensorgrams representing affinity data of mutant peptide binding to apanel of antibodies were assessed using multiple parameters. A visualinspection was first performed for each sensorgram to assess apparentmaximal response (“R_(max)”) relative to the wild-type PACAP peptide(1-38). Second, a visual inspection of the dissociation phase wasperformed with an emphasis on the curve shape relative to the wild-typePACAP peptide. Off-rates (dissociation rates) were calculated forwild-type PACAP peptide and the binding of each mutant peptide to thepanel of antibodies. Finally, as a control experiment to confirm theintegrity of each peptide variant (wild-type or mutant), the bindingaffinity of each member of the peptide library was individuallydetermined for each member of a panel of antibodies that were known tobind wild-type PACAP, to ensure that each Ala mutant PACAP peptideexhibited binding affinity that was similar to the binding affinity ofwild-type PACAP peptide. Collective assessment of all describedparameters identified PACAP amino acid residues important forPACAP/antibody binding.

Binding and dissociation data were obtained for binding of antibodiesAb1, Ab2, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab5, Ab7, Ab11,Ab12, Ab4, Ab3, Ab6, Ab8, Ab9, Ab22, and Ab23 to wild-type PACAP andPACAP mutants.

The PACAP residue positions contributing most to antibody binding wereinterpreted to jointly comprise the epitopes bound by each antibody.Based on data obtained in these alanine scanning studies, the epitopesbound by each antibody were concluded to comprise the followingresidues:

-   -   (i) Ab1: residues 5, 6, 8, 10, and 13 of human PACAP;    -   (ii) Ab2: residues 5, 6, 8, 9, 10, 13, and 14 of human PACAP;    -   (iii) Ab13: residues 6, 8, 9, 10, and 13 of human PACAP;    -   (iv) Ab14: residues 5, 6, 8, 9, 10, and 13 of human PACAP;    -   (v) Ab15: residues 5, 6, 8, 9, 10, 12, 13, and 14 of human        PACAP;    -   (vi) Ab16: residues 5, 6, 8, 10, and 13 of human PACAP;    -   (vii) Ab17: residues 5, 6, 8, 10, and 13 of human PACAP;    -   (viii) Ab18: residues 5, 6, 8, 9, 10, 12, and 13 of human PACAP;    -   (ix) Ab19: residues 4, 5, 6, 8, 9, 10, 12, 13, 14, and 17 of        human PACAP;    -   (x) Ab5: residues 3, 4, 5, 6, 7, 10, 13, and 14 of human PACAP;    -   (xi) Ab7: residues 6, 8, 10, 11, 13, 14, and 18 of human PACAP;    -   (xii) Ab11: residues 6, 8, 10, 11, 13, 14, 18, and 22 of human        PACAP;    -   (xiii) Ab12: residues 6, 8, 10, 11, 13, 14, and 18 of human        PACAP;    -   (xiv) Ab4: residues 8, 9, 10, 13, 14, 17, and 18 of human PACAP;    -   (xv) Ab3: residues 8, 9, 10, 11, 12, 13, 14, 17, and 21 of human        PACAP;    -   (xvi) Ab6: residues 5, 6, 9, 10, 12, 13, 14, and 17 of human        PACAP;    -   (xvii) Ab8: residues 7, 10, 13, and 14 of human PACAP;    -   (xviii) Ab9: residues 7, 10, 12, 13, 14, and 17 of human PACAP;    -   (xix) Ab22: residues 22, 23, 27, 28, and 31 of human PACAP; and    -   (xx) Ab23: residues 12, 20, 23, 24, 26, 27, and 28 of human        PACAP.

It was further noted based on the alanine scanning experimental resultsthat the affinity of each of antibodies Ab1, Ab2, Ab13, Ab14, Ab15,Ab16, Ab17, Ab18, Ab19, Ab5, Ab7, Ab11, Ab12, Ab4, Ab3, Ab6, Ab8, andAb9 for PACAP involves or depends on residues 10 and/or 13 of humanPACAP, and in some instances involves or further depends on residues 8and/or 14.

Additionally, it was observed that the affinity of each of antibodiesAb22 and Ab23 to PACAP involves or requires specific amino acid residuesthat are present in human wild-type PACAP38, but which are not presentin human wild-type PACAP27, e.g., residues 28 or 31 of PACAP38.

With respect to the foregoing alanine scanning results humanizedvariants of the subject anti-PACAP antibodies should interact with theidentical or substantially identical residues of human PACAP ashumanization should not appreciably impact the specificity of thebinding of the humanized anti-PACAP antibody to human PACAP compared tothe parent (unhumanized) antibody. Particularly, Ab3.H should interactwith the same residues on human PACAP as Ab3, Ab4.H should interact withthe same residues on human PACAP as Ab4, Ab5.H should interact with thesame residues on human PACAP as Ab5, Ab9.H should interact with the sameresidues on human PACAP Ab9, and Ab12.H should interact with the sameresidues on human PACAP as Ab12.

Antibodies which bind to the same or overlapping epitopes on human PACAPas the subject antibodies may be produced and identified using methoddescribed herein. It is reasonable to anticipate that antibodies whichbind to the same or overlapping epitope as any of the antibodiesidentified herein will likely possess similar biological activity absenta meaningful difference in binding kinetics. Particularly, suchantibodies should antagonize one or more of the biological effectselicited by PACAP analogously to the exemplified anti-PACAP antibodieswhich bind these epitopes. Additionally, antibodies that bind to thesesame or overlapping epitopes, or a subset of residues thereof, areanticipated to mimic the binding characteristics of the subjectantibodies. For example such antibodies are expected to selectively bindto PACAP and not bind or bind with much less affinity (weaker) to VIP orother peptides within this family of neuropeptides.

Having fully described and enabled the invention, the invention isfurther described by the claims that follow.

What is claimed is:
 1. A method of putatively determining whether anantibody or antigen binding fragment is suitable for use in treating orpreventing Pituitary Adenylate Cyclase-Activating Peptide(PACAP)-associated photophobia or light aversion, or precluding theonset of PACAP-associated photophobia or light aversion, in a humansubject in need thereof, which method comprises: (i) providing at leastone first non-human test subject and at least one non-human second testsubject; (ii) first administering PACAP to the at least one firstnon-human test subject and the at least one second non-human testsubject; (iii) subsequently administering to the at least one firstnon-human test subject who has been administered PACAP an anti-PACAPantibody or an antigen binding fragment thereof; (iv) comparing theresponse of the at least one first non-human test subject and at leastone second non-human test subject to light; and (v) if said first atleast one non-human test subject exhibits decreased light aversion ordecreased photophobia as compared with the at least one second non-humantest subject, identifying said antibody or antigen binding fragment asbeing putatively suitable for use in treating or preventingPACAP-associated photophobia or light aversion, or precluding the onsetof PACAP-associated photophobia or light aversion in a human subject inneed thereof; wherein said anti-PACAP antibody or antigen bindingfragment comprises an immunoglobulin variable heavy chain having theCDR1 sequence of SEQ ID NO: 44; a CDR2 sequence of SEQ ID NO: 46; and aCDR3 sequence of SEQ ID NO: 48; and/or an immunoglobulin variable lightchain having the CDR1 sequence of SEQ ID NO: 64; a CDR2 sequence of SEQID NO: 66; and a CDR3 sequence of SEQ ID NO:
 68. 2. A method ofscreening for an antibody or antigen binding fragment thereof suitablefor use in treating or preventing Pituitary Adenylate Cyclase-ActivatingPeptide (PACAP)-associated photophobia or light aversion, or precludingthe onset of PACAP-associated photophobia or light aversion, in a humansubject in need thereof, which comprises: (i) providing at least onefirst non-human test subject and at least one second non-human testsubject; (ii) first administering to the at least one first non-humantest subject an anti-PACAP antibody or an antigen binding fragmentthereof; (iii) subsequently administering PACAP to the at least onefirst non-human test subject and the at least one second non-human testsubject, wherein said first subject has previously been administeredsaid anti-PACAP antibody or an antigen binding fragment thereof; (iv)comparing the response of the at least one first non-human test subjectand at least one second non-human test subject to light; and (v) if saidfirst at least one non-human test subject exhibits decreased lightaversion or decreased photophobia as compared with the at least onesecond non-human test subject, identifying said antibody or antigenbinding fragment as being putatively suitable for use in treating orpreventing PACAP-associated photophobia or light aversion, or precludingthe onset of PACAP-associated photophobia or light aversion in a humansubject in need thereof; wherein said anti-PACAP antibody or antigenbinding fragment comprises an immunoglobulin variable heavy chain havingthe CDR1 sequence of SEQ ID NO: 44; a CDR2 sequence of SEQ ID NO: 46;and a CDR3 sequence of SEQ ID NO: 48; and/or an immunoglobulin variablelight chain having the CDR1 sequence of SEQ ID NO: 64; a CDR2 sequenceof SEQ ID NO: 66; and a CDR3 sequence of SEQ ID NO:
 68. 3. The method ofclaim 1 or 2, wherein the efficacy of said anti-PACAP antibody orantigen binding fragment thereof for putatively inhibitingPACAP-associated photophobia or light aversion, or precluding the onsetof PACAP-associated photophobia or light aversion in a human subject inneed thereof, is confirmed by administering said anti-PACAP antibody orantigen binding fragment to a human subject who comprises or chronicallyexperiences photophobia or light aversion caused by a chronic conditionassociated with photophobia or light aversion and determining in saidsubject whether said anti-PACAP antibody or antigen binding fragmentthereof inhibits PACAP-associated photophobia or light aversion, orprecludes the onset or the frequency of onset of PACAP-associatedphotophobia or light aversion in said human subject who comprises orchronically experiences photophobia or light aversion caused by acondition associated with photophobia or light aversion.
 4. The methodof claim 1 or 2, which comprises determining whether said anti-PACAPantibody or fragment thereof inhibits or diminishes light aversionbehavior in a first rodent administered PACAP and said anti-PACAPantibody or antigen binding fragment thereof, as compared to a secondrodent administered PACAP which is not administered said anti-PACAPantibody or antigen binding fragment thereof.
 5. The assay of claim 1 or2 wherein said anti-PACAP antibody is full-length.
 6. The assay of claim1 or 2 wherein said anti-PACAP antibody is humanized.
 7. The assay ofclaim 1 or 2 wherein said anti-PACAP antibody comprises a human IgG1,IgG2, IgG3 or IgG4 constant region.
 8. The assay of claim 1 or 2 whereinsaid anti-PACAP antibody comprises a human IgG1 constant region.
 9. Theassay of claim 1 or 2 wherein said anti-PACAP antibody isnon-glycosylated.